We describe a new species of bat in the genus Myotis (Vespertilionidae, Myotinae) from the coastal mountains of Venezuela. The new species (Myotis handleyi, sp. nov.) can be distinguished from other South American congeners by the following set of traits: dorsal fur long, silky, and bicolored with burnished tips; skull long; rostrum long and broad; frontals moderately to steeply sloping; sagittal crest absent or very low; plagiopatagium broadly attached to the foot at the level of the base of the toes; fringe of hairs along the trailing edge of uropatagium absent; and fur on uropatagium not reaching knees. We review Colombian and Venezuelan samples of Myotis, covering all of the currently recognized species known from these countries. Based on our analyses, we provide a sketch of the taxonomic diversity of the genus in Colombia and Venezuela, along with a key to their identification. Among other conclusions, we elevate M. nigricans caucensis to the species level; confirm that populations of M. nigricans from the opposite sides of the Andes represent the same taxon; retain J.A. Allen's names M. esmeraldae, M. bondae, and M. maripensis in the synonymy of M. nigricans; and document clinal variation in size along an altitudinal gradient for M. nigricans, with larger specimens from higher elevations. This research, based on museum and field collections, is one of a series of studies by the senior author re-evaluating species limits among Neotropical Myotis.