The Jamaican boa ( Epicrates subflavus ; also called Yellow boa) is an endemic species whose natural populations greatly and constantly declined since the late 19th century, mainly because of predation by introduced species, human persecution, and habitat destruction. In‐situ conservation of the Jamaican boa is seriously hindered by the lack of information on demographic and ecological parameters as well as by a poor understanding of the population structure and species distribution in the wild. Here, using nine nuclear microsatellite loci and a fragment of the mitochondrial cytochrome b gene from 87 wild‐born individuals, we present the first molecular genetic analyses focusing on the diversity and structure of the natural populations of the Jamaican boa. A model‐based clustering analysis of multilocus microsatellite genotypes identifies three groups that are also significantly differentiated on the basis of F ‐statistics. Similarly, haplotypic network reconstruction methods applied on the cytochrome b haplotypes isolated here identify two well‐differentiated haplogroups separated by four to six fixed mutations. Bayesian and metaGA analyses of the mitochondrial data set combined with sequences from other Boidae species indicate that rooting of the haplotypic network occurs most likely between the two defined haplogroups. Both analyses (based on nuclear and mitochondrial markers) underline an Eastern vs. (Western + Central) pattern of differentiation in agreement with geological data and patterns of differentiation uncovered in other vertebrate and invertebrate Jamaican species. Our results provide important insights for improving management of ex‐situ captive populations and for guiding the development of proper in‐situ species survival and habitat management plans for this spectacular, yet poorly known and vulnerable, snake.