Evolutionary relationships among bats of the genus Rhogeessa (Chiroptera: Vespertilionidae) are poorly understood because of the morphological similarity of many of the species and the limited resolution of karyotypes and allozymes in previous studies. Previous karyotypic studies reported several populations that differ by Robertsonian centric fusions, which led to a proposed mechanism of speciation called speciation by monobrachial centric fusions. Here, we present a molecular phylogenetic analysis of 8 of the 10 currently recognized species of Rhogeessa using the mitochondrial DNA gene cytochrome b as well as new karyotypic data. The results are generally consistent with speciation by monobrachial centric fusions because karyotypically distinct populations typically comprise monophyletic maternal lineages. One exception was 2 individuals that were possible hybrids between R. tumida (2n = 34) and R. aeneus (2n = 32). We found ostensible species-level differentiation among 3 karyotypically identical (2n = 34) but geographically separated populations of R. tumida. Examination of new karyotypic data shows a population from western Ecuador to have 2n = 42 and study of molecular data shows it to be phylogenetically distinct from both the karyotypically identical R. genowaysi from Mexico and the South American R. io (2n = 30) to which it was previously allocated. We recognize this population by its available name, R. velilla. We also found an unexpectedly close relationship between Baeodon alleni and R. gracilis.