The greater mouse-tailed bat (Rhinopoma microphyllum) possesses a large geographical range, covering most of the arid and warm areas of the Old World. We studied the genetic variability of this species using two mitochondrial markers (the cytochrome b gene and the control region), from several Israeli colonies and from over most of the species' range. Our results show that the cytochrome b sequences, unlike those of the control region, are too conserved to separate among R. microphyllum populations. Based on the control region sequences, a high level of sequence similarity was found within the Israeli population. Three clades were observed over the species' range: Oriental, Intermediate and Palaearctic. This division supports most of the traditional taxonomy of the species. The Israeli population, which belongs to the Palaearctic clade, was found to be isolated from the Oriental and Intermediate clades. We suggest that the colonization of the greater mouse-tailed bat in the Levant occurred from African populations during the late Pleistocene, when many Saharan plants and animals penetrated the northern part of the Great Rift Valley.