This article provides a phylogenetic analysis of five nuclear and mitochondrial cytochrome b genes of palaearctic serotines. Nuclear data yield five monophyletic clades: Botta’s serotine and the South African long-tailed house bat E. hottentottus; the common serotine bat (including all studied E. serotinus subspecies and andersoni form of undefined status) and the meridional serotine E. isabellinus; the Gobi serotine, including Bobrinski’s serotine; the northern bat; and New World serotines. We found latest taxonomic decisions regarding mirza and pachyomus questionable and needing further revision. The significant inconsistency between mitochondrial and nuclear phylogenies obtained for genes of different inheritance systems suggests repetitive introgression events in the evolution of the genus.