Species with restricted geographic distributions consisting of isolated populations are particularly susceptible to extinction because these demes face an increased risk of disappearing due to environmental, demographic, and genetic stochasticity. We used partial DNA sequences of the cytochrome b (1083 bp) and ND2 (1041 bp) mitochondrial genes to investigate the phylogeography and population genetics of Anolis cooki, a threatened lizard endemic to the southwestern coast of the Caribbean Island of Puerto Rico. Maximum likelihood and Bayesian methods revealed relatively shallow genetic differentiation among 27 unique haplotypes (from 52 individuals) from the known extant populations of A. cooki in mainland Puerto Rico. Despite this pattern, specimens from the same geographic area tended to nest together. The most basal division within A. cooki is between haplotypes from the three westernmost populations (Punta Águila, Morrillos, Playa Santa) and the remainder demes (Bahía Ballena, La Cueva, Punta Verraco). The three westernmost populations of A. cooki are separated from their conspecific demes by the Guánica Bay and the Loco River drainage system, which together may represent a physiographic barrier for A. cooki. Each population of A. cooki only has private haplotypes; in other words, there are no shared mitochondrial types between populations. Because the number of private haplotypes can be used as an indirect measure of gene flow, this finding suggests that currently there is no migration among demes, and that each is an independent demographic unit, despite the relatively short distances (ca. 2 km) that separate some of them. Pairwise F ST values and spatial analyses of molecular variation confirmed the existence of distinct groups of genetically defined sampling areas, and of significant molecular variation among populations within groups and within populations. The conservation status of the populations of A. cooki varies greatly. The demes from Punta Águila, Morrillos, and Bahía Ballena inhabit protected areas, and are larger, genetically diverse, and seemingly stable. The population from Playa Santa showed a high level of genetic diversity, but it occurs in an area that has been intensively developed for residential and touristic purposes, and its long-term survival is uncertain. A. cooki is also known from Caja de Muertos, an island off the southcentral coast of Puerto Rico. Surveys conducted on September 2006 and March 2007 did not produce any specimens, and a thorough assessment of Caja de Muertos is needed to determine the present status of A. cooki on the island.