Biodiversity and wild populations are globally threatened by a wide range of actors. The genus Iguana, widely distributed throughout the Americas, is under threat by invasive species, hybridization, the global pet trade, and habitat destruction. This holds especially true for the insular lineages, with the Critically Endangered I. delicatissima having experienced a > 75% range decrease, primarily through hybridization with non-native iguanas. We collated published microsatellite data and genotyped samples from new localities to construct a distribution-wide Iguana dataset built from 190 individuals for 17 microsatellite loci. This enabled us to identify patterns of genetic differentiation within and among populations, and identify key loci and private alleles for use in conservation management. Our analyses reveal clear separation between I. delicatissima and the I. iguana complex, highlighting the power of eight key microsatellite loci for the study of hybridization dynamics. Genetic differentiation within I. delicatissima identifies four clusters that aid decision making for conservation management action. Within the I. iguana complex, we increase mainland localities by 11-fold and recover 3.5 × more alleles across all loci than previously known. Overall, we identify 112 (48% private) and 76 (25% private) alleles for mainland and island lineages, respectively. We highlight loci sets to identify (1) non-native or hybrid iguanas in insular populations and their genetic origin, and (2) genetic origin of insular iguanas in the global pet trade. Overall, we provide a reference for Iguana microsatellite loci in order to allow standardization and comparison among studies, aiding broader assessment of research and conservation hypotheses. We thank Ty Park and all donators to IguanaFest 2019 for financial support. Peer reviewed