Abstract The genus Myotis comprises a diverse group of vespertilionid bats with worldwide distribution. Twenty-eight Neotropical species are currently recognized. In this study, we evaluate molecular and morphological variation in the M. keaysi complex, a high elevation clade of Neotropical myotine bats characterized by complex taxonomy and high morphological variation. A phylogeny inferred with cytochrome-b sequences recovered two clades composed of samples traditionally assigned to M. keaysi, with 9% of genetic divergence between them. These clades were also suggested as putative distinct species by molecular species delimitation methods. Qualitative and quantitative morphological analyses indicated a phenotypic discontinuity between specimens from central Andes (including the holotype of M. keaysi) and western lowlands of Ecuador, showing strong congruence between molecular and morphological approaches. We therefore describe a new species for the Tumbes-Choco-Magdalena region, documenting their external and cranial diagnostic characters by comparing them with other Neotropical species. In addition, we provide an emended diagnosis for our new concept of M. keaysi.