Qualitative and quantitative variation in morphology was assessed for 6 species of Myotis from South America to determine which taxa occur in Paraguay, characteristics that allow for their discrimination, and the degree of geographic and secondary sexual variation that occurs in Paraguayan forms. Secondary sexual dimorphism and geographic variation were evaluated from univariate and multivariate perspectives. Multivariate morphometric differences in cranial morphology among taxa, independent of size, and mensural characters that best separate species were determined using principal components analysis followed by size-adjusted discriminant function analysis. Five species, M. albescens, M. nigricans, M. riparius, M. ruber, and M. simus, occur in Paraguay and can be distinguished using combinations of cranial and external characteristics. No significant sexual dimorphism or geographic variation was detected in these taxa of Myotis in Paraguay. M. levis has not been recorded for Paraguay as previously reported, but it may occur in the country. M. simus, a species previously considered to be restricted to the Amazon Basin, occurs as far south as Paraguay and northern Argentina. Although M. simus varies significantly in size across its range, highly differentiated cranial and external morphology allows for recognition of this species across its range.