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The vespertilionid bat genus Hesperoptenus, comprising only five species and showing a distribution in the tropical regions from India to Sulawesi, was hitherto underrepresented in recent taxonomic studies. Here, modern cytogenetic techniques were applied to compare the karyotypes of two Hesperoptenus species, H. tomesi and H. blanfordi. Despite having the same chromosome number, 2n = 32, the karyotypes of H. tomesi and H. blanfordi were found to differ extensively. Instead, H. tomesi was found to share all nine derived Robertsonian fusion products with the previously published karyotype of H. doriae (2n = 26), which points to a closer relationship between both species. In line with our chromosomal data, mitochondrial DNA analyses also provided evidence for a closer relationship of H. tomesi with H. doriae and H. tickelli, for which no banded karyotype has been examined. Again, H. blanfordi was clearly separated from the remaining Hesperoptenus species. As a consequence, the subgenus Milithronycteris, comprising all species of Hesperoptenus except the type species H. doriae, is rendered paraphyletic. We therefore suggest to synonymize Milthronycteris with the subgenus Hesperoptenus. A chromosomal character, found in all three Hesperoptenus species examined but absent in other vespertilionid genera studied so far, namely a particular G-banding pattern on the homolog to Myotis chromosome MMY13, may serve as a synapomorphy for the genus Hesperoptenus. [ABSTRACT FROM AUTHOR]