Histiotus is a Neotropical genus of bat that currently includes 11 species. The systematics of Histiotus has been the focus of several studies over the last decades. However, no broad systematic revision has been made, and taxonomic issues such as synonymies, use of subspecies, and specimens that do not fit the description of valid species still persist, as pointed out by several authors. Histiotus alienus was described in 1916 and is known only by the holotype. Here we present a second record of H. alienus and an amended diagnosis of this species. We use qualitative, quantitative, and morphometric analyses based on data from 184 specimens of Histiotus and almost all valid species. Our amended diagnosis establishes the taxonomic limits of H. alienus, as well as a comprehensive comparison with congeners. We also explore new diagnostic characters for H. alienus and provide a few notes on the natural history of this species. Our results highlight skull similarities among Histiotus species and reinforce the usefulness of external morphology for their correct identification. Despite our new insights into the taxonomy of the genus, several taxonomic issues remain, and a comprehensive revision of the genus is needed.