Rock-iguanas of the genus Cyclura are the largest native terrestrial herbivores of the West Indies. Most species are currently under a high risk of extinction. In order to assess genetic variation in Cuban rock iguanas (Cyclura nubila nubila), we sequenced a fragment of mitochondrial DNA (894 bp long region including a part of the ND4 subunit of the NADH gene and the tRNA genes histidine, serin and leucin (partial)) in 21 iguanas from European Zoos and private breeders. The animals sampled represent founders and important representatives of the studbook population. The data was combined with published sequences of other species/subspecies of the genus and outgroups. We used neighbour joining, maximum likelihood, maximum parsimony and Bayesian methods to reconstruct the phylogenetic tree. The results are congruent with the previously reported paraphyletic relationship of C. nubila with respect to C. cychlura from the Bahamas. Surprisingly, within C. n. nubila, we found previously undescribed haplotypes phylogenetically closer to those of the subspecies C. nubila caymanensis from Little Cayman Island, or C. nubila lewisi from Grand Cayman Island. Thus, our results show that C. n. nubila as currently defined represents more distinct lineages and is therefore a composite taxon. Nevertheless, as rapidly evolving isolates these taxa are still worthy of conservation efforts. We hypothesize that the basal divergence of the C. nubila–C. cychlura clade occurred in Cuba and the above mentioned taxa from the neighbouring islands are a result of relatively recent over-water dispersal. In conclusion, our data suggest that special attention should be devoted to conserving Cuban rock-iguanas.