Geoffroy's bat, Myotis emarginatus, is the only species of the African clade of the genus Myotis distributed in the south-western part of the Palaearctic. Due to its extensive distribution range, stretching across several ecologic zones from the European and African Mediterranean, Central Europe, through the Levant and Caucasus to West Turkestan and south-eastern Middle East, this bat is traditionally considered to be a variable and polytypic species. While one subspecies was recognized in Europe and North Africa, up to four subspecies were reported from the Asian part of the species range. Nevertheless, the systematic positions of different populations and the validity of particular taxa remained unclear. Our aim was to revise the phylogenetic status of M. emarginatus and, for the first time, genetically analyse samples from the Asian part of its range to provide new insight into its intraspecific variation. We analysed sequences of two mitochondrial and three nuclear markers from more than 130 samples from all parts of the species range, together with sequences from other species from the African clade of the genus Myotis. According to the previous morphometric results of body and skull dimensions, M. emarginatus can be divided into two groups of populations: the small-sized and more variable bats of Europe, the Maghreb and Levant; and the large-sized bats of the rest of the Asian range. This division was well supported by mitochondrial genes, which separated two main lineages within the species: the western lineage from Europe, the Maghreb and Levant; and the eastern lineage from the eastern Middle East and West Turkestan. Both mitochondrial lineages were further divided into two sublineages: the western lineage to sublineages from the Holy Land and the rest of the Mediterranean range; and the eastern lineage to sublineages from Oman and southern Iran, and northern Iran and West Turkestan. In contrast, the nuclear genes reconstructed only one lineage through the whole distribution range, suggesting M. emarginatus to be a monophyletic species. Nevertheless, on the basis of previously described geographical variability in morphology and the newly described mitochondrial variation, we recognize two subspecies within M. emarginatus: small-sized M. e. emarginatus distributed in the Mediterranean, western and central Europe and Levant; and large-sized M. e. desertorum in the eastern Middle East, from Oman to West Turkestan.