Myotis nesopolus currently comprises two subspecies. The nominate subspecies (M. n. nesopolus) occurs on the Caribbean islands of Curaçao and Bonaire, Netherlands Antilles, whereas M. n. larensis is known from mainland South America in northeastern Colombia and northwestern Venezuela. Our Maximum Likelihood phylogenetic analyses of cytochrome-b gene sequences recovered M. nesopolus as a paraphyletic group, with M. n. nesopolus and M. n. larensis as non-sister lineages. The haplotype network indicates that these two subspecies do not share any haplotypes and are in different evolutionary trajectories. Additionally, these two subspecies can be distinguished on the basis of qualitative and quantitative morphological traits. This pattern supports the recognition of M. nesopolus and M. larensis as full species. Our results also reveal that the assemblage of Caribbean Myotis do not form a monophyletic group. Caribbean species are phylogenetically close to mainland species from northern South America and Central America, suggesting that colonization of Caribbean islands happened multiple times.