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Currently, four species of Myotis are known from the islands of the Caribbean (Myotis dominicensis, M. martiniquensis, M. nesopolus, and M. nigricans). Myotis dominicensis and M. martiniquensis are endemic to the Lesser Antilles, whereas M. nesopolus and M. nigricans are considered conspecific with mainland populations. Recent phylogenetic and phylogeographic studies provided hypotheses regarding the origin and diversification of M. dominicensis and M. martiniquensis. However, these studies focused primarily on convergent morphology or distribution patterns of this genus and not on the evolutionary history of Caribbean Myotis. Here, we explore variation across multiple datasets generated from Caribbean Myotis. We present morphologic and genetic (mitochondrial and nuclear) data from an extensive sample of Caribbean Myotis species, including the previously unsampled taxa M. martiniquensis nyctor and M. nesopolus. Our data indicate that the historically recognized subspecies M. m. nyctor is genetically and morphologically distinct from M. martiniquensis, warranting recognition of a third Caribbean endemic—Myotis nyctor. Moreover, we provide evidence of unrecognized species-level variation in Caribbean and northern South American populations of Myotis.