Nucleotide sequences of the mitochondrial cytochrome b gene are reported from bats of the genus Myotis including species of the endemic southern African subgenus Cistugo, Myotis (Cistugo) sebrai and Myotis (Cistugo) lesueuri. We also examined Myotis annectans from Southeast Asia, and Myotis macropus from Australia. The two species of Cistugo and Myotis annectans represent the only species of Myotis to differ in chromosome number from the common 2n=44 found in >40 species. Our results show that the two species of Cistugo are more divergent from the other species of Myotis than several other well-recognized genera and we recommend elevating Cistugo to full generic rank. Myotis annectans groups well within Myotis, clustering with other Southeast Asian and Japanese species, and thus represents the only species of Myotis known to have diverged from the common 2n=44 karyotype. Myotis macropus clusters within a clade that includes Southeast Asian species.