For species living in naturally fragmented habitats, the identification of conservation units is particularly challenging. Turks and Caicos Rock Iguanas, Cyclura carinata, are endemic to the Turks and Caicos Islands (TCI). These Critically Endangered lizards inhabit less than 5% of their historic range. A complete study of the geographic distribution of genetic variation is now particularly important for this species due to the onset of translocation programs. To fill this knowledge gap, we genotyped 280 individuals from 30 sampling locations across the species range were genotyped at 29 microsatellite loci, and variation within the mitochondrial ND4 gene was assessed by sequencing, or the use of PCR-RFLPs. A protein profiling analysis of femoral pore secretions was also conducted on a subset of samples. Microsatellites suggest that a recent, common population ancestry, or a simple reduction in gene flow, cannot account for the significant differences detected between a western and an eastern lineage within the TCI (F-ct = 0.202, p ae0.01). The same pattern was confirmed by mitochondrial DNA sequence, significantly distinguishing between the western and eastern haplotypes (I broken vertical bar(ct) = 0.96, p ae0.01). Protein profiling provides further support, differentiating a western and eastern lineage. Implications of these results are that at least two evolutionarily significant units (ESUs) can be recognized across this taxon's geographic range. Multiple management units, within each ESU, can also be defined. Future management planning for C. carinata should consider these evolutionarily independent lineages.