The genus Myotis (Vespertilionidae, Myotinae) comprises a diverse group of small to large-sized vespertilionid bats that present a worldwide distribution. Twelve South American species are currently recognized. In this paper we evaluate the morphological and morphometric variation observed in South American populations of the most widespread species, Myotis nigricans. Against this background, two forms can be morphologically distinguished from M. nigricans and other known South American species. We describe these new species, documenting their diagnostic external and cranial characters by comparing them to other sympatric and cryptic species of South American Myotis. In addition, we provide an emended diagnosis of Myotis nigricans.