Several characters in With Pearl and Ruby Glowing are interpreted as transgender when they were not written as such in the original source material. In many cases, this contributed to their abuse. It's also a frequent means to explain male characters giving birth to children or female characters fathering them.
- Mr. Willard Tweedy is now Ms. Wilhelmina Tweedy, having come out as trans after leaving her abusive wife, who sexually assaulted her after learning she was trans and gave birth to the resulting child in secret after Wilhelmina ran away.
- Julien is a transgender man, playing off the fact that lemurs are matriarchal in real life. He was raped by a religious cult due to being trans, impregnating him with Mort, who is his child in this universe.
- Riley is nonbinary in this universe, though they aren't out to their classmates. This is based on a popular headcanon meant to explain Riley having mixed-gender emotions. Ratigan exploited their insecurity about their gender to assault them.
- Dr. Flug is a trans man, with 5.0.5. being his human son conceived after Ratigan raped him.
- Kenai is a trans man; he was taken by the Ark and forced to have sex with Koda.
- Ruby and Sapphire, in canon, are both genderless female-presenting aliens. In this fic, they are a cis woman and a trans woman, though it's kept ambiguous which woman is which; this allows Garnet to be their biological child. The other Gems appear to all be female-identified. Rose, Lapis, Peridot, and Ocean Jasper are confirmed DFAB by having given birth; Peridot is Lapis' mother, Rose died in childbirth with Steven, Lapis gave birth to Malachite, and Ocean Jasper gave birth to Zebra Jasper. Malachite and Zebra Jasper were/are DFAB babies, with Malachite dying a few days after birth and Zebra Jasper not old enough to state whether their identity may be different. Jasper is Malachite's and Zebra Jasper's intersex father who identifies as female and was assigned male at birth, but doesn't consider herself trans, just someone who her mother forced to crossdress. She confirms at least one or two of the Famethyst have the same condition, specifically including Lace Amethyst. It's speculated but not confirmed that said condition is PAIS, which can cause a feminine appearance with fertile sperm. Holly Blue is the birth mother of Jasper and the Famethyst. Emerald is nonbinary and DMAB (Spinel, who was raped by them, reports that Emerald has a penis), and works as a drag queen like their voice actor. Amethyst is genderfluid, saying that she's a demigirl and sometimes a demiboy. Nice Lapis is a trans girl. Steven himself is a trans boy with "Nora" (the name his parents considered for him if he were a girl) being his dead name. Pearl is a trans woman.
- Mew and Mewtwo are respectively a cis girl and cis boy instead of being genderless. Meowth is a trans girl, in honour of the canon Meowth's voice actress Maddie Blaustein. Bea is nonbinary and uses it/its pronouns as a Shout-Out to a mistranslated wiki. Sylveon is a human trans woman who never got to transition officially but explained her identity in letters to her cis sister Eevee. Hilda and Hilbert are a Composite Character trans girl, with Hilda being her chosen name and Hilbert her deadname, while Nate and Rosa are similarly composited into a cis boy who gets Dragged into Drag. Ash's Pikachu is a female pet chinchilla. The player character of Magikarp Jump is nonbinary. The Mimikyu and Scraggy from Pokétoon are cis-female and nonbinary respectively.
- Pleakley is a trans woman as opposed to a Wholesome Crossdresser.
- Lynn Loud Jr. is a trans girl, but Foxy Loxy and her friends have parents that are members of God's Will First, so they assault her for it. Lana is questioning her gender.
- Flynn Rider is a trans man; the corrupt cops that assaulted him called him "Princess" because of this.
- Francis is trans and was subject to transphobic remarks from the flies at the bar because his face still looked lady-like.
- Peekablue is a trans boy and was attacked by God's Will First after Flutterina encourages Glimmer to make a False Rape Accusation against him, unaware that he was trans.
- Muscle Man is trans according to the "Parks and Rec" chapter, which mentions that his chest is unbound. Margaret is a trans woman as a reference to her canon species having male plumage.
- All of JNPR are binary trans, with Jaune and Ren being trans boys and Nora and Pyrrha being trans girls. Also, Penny Polendina and Ruby Rose are trans girls.
- Auntie Uncle is non-binary rather than "Uncle" being a first name, and uses the Spivak ey/em/eir pronouns.
- Sailor Uranus is a Bokukko canonically described as a blend of man and woman; here, he's specifically a bigender he/him lesbian. Zoisite is a trans girl as a reference to her She's a Man in Japan status.
- Mr. Baldwin is trans and pregnant (willingly, unlike most cases in the fic) as a reference to him being a male seahorse in the show.
- Elina is a trans woman, and Erika Juno's gender is described as "none gender with left fluid
". Laverna pretends to be trans online, but isn't really. - Steele is a trans man who gets pregnant after drunkenly raping Balto.
- Shaun is a trans boy. In "Coming Out", he came out to a still closeted Wilhelmina, who told him that he could stay with her and Wallace if his family didn't accept him.
- Stacie is a trans girl, being a Composite Character of Todd, Tutti, and her canon self.
- Zipp Storm is trans due to her Tomboyish Voice in canon, as well as for plot reasons.
- Perky, who was in canon a female peacock, is a hijra/trans woman here.
- Daffy Duck is depicted as very apathetic towards gender in general and testing out different pronouns.
- Pinocchio, Emira, Meilin Lee and Peppa Pig are cisgender intersex. Pinocchio has genital agenesis and his assigned gender was determined through a blood test, while Emira has Turner Syndrome, giving her a DFAB appearance. Meilin is undergoing contrasexual puberty as an analogue to her canon panda transformation, and is keeping her beard. Peppa Pig has PCOS, leading her to be put on puberty blockers after she began puberty at only 4 years old. ENA is also intersex.
- Beth Lestrade is a trans woman.
- Bidoof's trainer is nonbinary, using xe/xem/xir pronouns. Bidoof was female in the original short, judging by her tail shape, and is a girl here too, but Lucario doesn't have dimorphic traits in the cartoon, while here he's confirmed as a boy.
- Michael Collins uses he/she/they pronouns and is later revealed to be nonbinary.
- Kanamori is canonically DMAB femme, but a specific identity and English pronouns haven't been stated officially; here they're a she/they trans woman.
- Angel Dust is a cis man, but with what he describes as a hormone problem which gives him breasts instead of his canonical puffy chest fur.
- While Monkie Kid isn't canonically trans, he's a demiboy here, using he/they pronouns. Red Son is canonically nonbinary, but uses he/him pronouns in the show and the Mandarin pronouns ta/ta de in the fic.
- Lake is nonbinary and uses co/cos pronouns. Ryan is also nonbinary and uses they/them pronouns.
- Lenore is a trans man who goes by Lenny, his fake mustache a reference to the one stolen from the canon material.
- Natsuki is a trans girl, and according to Monika she has been molested by her father ever since she came out.
- Homestar and Marzipan are both trans, and Marzipan knocked up Homestar. The Cheat is non-binary and uses they/them pronouns whereas in the original source material, The Cheat's pronouns are he/him.
- Donny is a trans boy, as an analogue for his desire to be a unicorn. Bliss identifies as female as in canon and is able to get pregnant, but is intersex with ovotesticular disorder, was assigned male at birth, and chose to keep her masculine first name, Biswanath. Indian culture classifies her as hijra. Snake is nonbinary. Brick is a trans girl, Butch is nonbinary, and Boomer is a trans boy.
- Rainbow Mane is a trans woman, in reference to how she was voiced by a male actor.
- Officer Fox is a trans woman as a reference to how she was originally male in the manhwa Brave Hedgehog.
- The boy and girl from Skies Forever Blue —here named Taffy and Skye respectively— are trans, their deadnames being Hana and Toby as a reference to the video’s creators.
- Harry Potter is a trans boy, which lead to his transphobic teacher Umbridge forcing him to carve lines into the back of his hand. Hermione has also been confirmed to be a trans girl and the Weasleys all have a hereditary intersex condition. Dumbledore is a drag queen.
- Glinda is either a trans woman or a drag queen or somewhere in between - the distinction wasn't made as clear in 1969, when the scene featuring her is set.
- Morticia and Gomez are both trans; we see they received a card at Wednesday's birth reading IT'S PROBABLY A GIRL.
- Nimona is non-binary and goes by ne/nir/nem pronouns whereas in the original movie and graphic novel, Nimona's pronouns are she/her.
- Yasuho Hirose, a Composite Character with Koichi, is a trans woman. Jolyne Cujoh is a trans man (as a reference to the transmasculine prisoner in the beginning of Stone Ocean) and is now named Joel. Dragona Joestar was gender ambiguous in Jojolands, but here is nonbinary, specifically, genderfuck.
- Dolly and Dizzy are dilbaa - a Navajo-specific identity describing AFAB people with a masculine gender expression. By Western standards, they're butch/tomboyish cis girls, though the term can also cover trans men.
- Miss Tarantula is a trans woman which got her sent to a male prison when the gang was arrested.
- Talulah Robinson is a trans woman.
- Averted with Wolfie, who still identifies as male even though he prefers to wear traditionally feminine clothes, and was misdiagnosed as trans when he was younger.
- Smurfette is a trans woman.
- Little Rural Riding Hood was a Lhamana, a Zuni-specific term for transfeminine people.
- Bluey, Bingo, and Muffin identify as brotherboys, an Australian Aboriginal-specific term for DFAB people with masculine identities. Muffin specifically appears to identify as female in most ways except for wanting her penis back, as she's intersex and was operated on in infancy. Alfie is mentioned to be a sistergirl, the analogous term for feminine DMAB people.
- Iris is a trans girl.
- Starcatcher is a trans woman.
- The prince, Sparkles' dad, is a trans man, who gave birth to her prior to coming out, being divorced by his husband and marrying the witch, Gloom's mother.
- Re-Verse Plagg, Shadybug and Claw Noir are trans girls.
- Dexter Charming is a trans boy.
- In a reference to the cartoon causing Viewer Gender Confusion, Keyla is mentioned to have been a trans man. Deyna is a sistergirl while Nimbalo is a brotherboy. Also in reference to the cartoon making Pallum female, here Pallum is described as androgynous and referred to with they/them pronouns in the narration, later mentioning that they're intersex (specifically DMAB but never having had a typical puberty).
- Voyd is a trans woman, which actually plays a fairly big role in her story as it allows her to prove she was framed. Context implies that so is Brick.
- The Australian Olympics mascot Fatso the wombat is a woman.
- Ash Fox is genderqueer and uses he/she/they pronouns.
- Averted with Strawberry Crepe Cookie, who calls themself a "girl" but is confirmed by the writers to still be nonbinary but closeted in the fic, as they're living in the UAE where being out isn't safe. Played straight with Werewolf Cookie, who's a closeted trans woman.
- Jade Harley is undergoing masculine puberty due to being intersex and on hormone replacement therapy. John is genderfluid. Fiamet is a trans girl.
- Frylock is genderfluid, with them using he/she/they pronouns and Meatwad is gendervoid and uses he/they pronouns.
- Doofenshmirtz is a trans man who had Vanessa with a donor. Norm is a trans woman whose name here is Norma, and is Doof's ex-wife Charlene's bio daughter via a sperm donation from Doof's brother Roger, raised as Doof's child.
- Both Black Spy and White Spy are trans women, as well as being Composite Characters with Plague Doctor and Plague Nurse.
- The kite girl from One By One is a trans girl, who is forced to be closeted after being sent to a male prison.
- Mme Upanova, Hyacinth Hippo, and Elephanchine Elephant are cis men Dragged into Drag as part of the Numbers Gang hierarchy.
- Tweak is a he/she lesbian and Kwazii is genderfluid, as references to the books having typos with their pronouns.
- Jeffrey is a trans girl called Desiree. Somewhat downplayed, given that she would have canonically been this, if not for some Out of Order episodes and her series being cancelled.
- Pickles the Drummer and Toki Wartooth are both trans men here.
- Ruby and Rock are both trans here.
- Pepper is intersex and nonbinary, specifically described by the authors as a theybie (a child intentionally raised gender-neutral from the beginning, in some cases and presumably this one because they're intersex).
- Sarah and Rachel are both trans women.
- Herbert P. Bear is a post-op trans man. Rookie is trans and is now pregnant with Jet Pack Guy's child. DJ Cadence and G Billy are a trans woman and trans man respectively.
- Bowser is a trans man and Luigi is genderqueer.
- Miss Piggy is a trans woman. Gonzo is on the genderqueer spectrum.
- Stitch and Mo are a trans boy and a trans girl respectively.
- Pluey is a sistergirl and Battat is a trans man.
- Road Runner is a demiboy and uses he/they pronouns.
- Penny is a trans woman.