Phylogenetic relationships and species delimitation in African Vespertilionini have been a long-standing subject of debate and are still controversial, although recent molecular analyses have shed light onto some of these issues. In this study we employed a comparative cytogenetics approach for the delineation of chromosomal homology and for the detection of shared chromosomal characters, which were then used to support proposed phylogenetic relationships. Here, we present karyotype analyses of five African Vespertilionini, Laephotis kirinyaga (2n = 32), Neoromicia guineensis (2n = 26), Pseudoromicia brunnea (2n = 36), Nycticeinops happoldorum (2n = 24), and Nycticeinops schlieffenii (2n = 34), which were complemented by mitochondrial DNA sequence analysis for species classification of all our specimens. Our cytogenetic analyses revealed that three derived Robertsonian fusion chromosomes, 7/11, 8/9, and 10/12, characterize the karyotypes of four African Vespertilionini genera, i.e. Laephotis, Neoromicia, Afronycteris, and Pseudoromicia, for which we propose to constitute a new subtribe, Laephotina. A rare chromosomal rearrangement, an X-autosome translocation, was found in the studied N. guineensis female. The genus Nycticeinops is characterized by a high intrageneric karyotype diversity. In only two of all four analyzed species, i.e. N. happoldorum and N. crassulus, a common chromosomal feature, the fusion product 1/13 was detected. Further, for the recently described East African serotine, L. kirinyaga, we present the second record for West Africa. The cytochrome b sequence of our N. guineensis specimen from Ivory Coast showed 4% divergence to that of its closest relative, N. somalica from Kenya.