The Lesser Antillean Iguana (Iguana delicatissima) is an endangered species threatened by habitat loss and hybridization with non-native Green Iguanas (Iguana iguana). Iguana delicatissima has been extirpated on several islands, and the Green Iguana has invaded most islands with extant populations. Information is essential to protect this species from extinction. We collected data on 293 iguanas including 17 juveniles from St. Eustasius, one of the few remaining I. delicatissima strongholds. Genetic data were leveraged to test for hybridization presence with the Green Iguana using both mitochondrial and nuclear genes, including 16 microsatellite loci. The microsatellites were also analyzed to estimate genetic diversity, population structure, and effective population size. Using molecular and morphological data, we identified 286 I. delicatissima individuals captured during our first fieldwork effort, and 7 non-native iguanas captured during a second effort, showing hybridization occurs within this population. Comparing homologous microsatellites used in studies on Dominica and Chancel, the I. delicatissima population on St. Eustatius has extremely low genetic diversity (HO = 0.051; HE = 0.057), suggesting this population is genetically depauperate. Furthermore, there is significant evidence for inbreeding (FIS = 0.12) and weak spatial genetic structure (FST = 0.021, P = 0.002) within this population. Besides immediate threats including hybridization, this population's low genetic diversity, presence of physiological abnormalities and low recruitment could indicate presence of inbreeding depression that threatens its long-term survival. We conclude there is a continued region-wide threat to I. delicatissima and highlight the need for immediate conservation action to stop the continuing spread of Green Iguanas and to eliminate hybridization from St. Eustatius.