Small mammals of genus Mastomys are important reservoir hosts of many diseases affecting humans.Knowledge about taxonomic status and distribution range of members of the genus Mastomys in southern Africa is of great importance.Study methods included skull morphometrics (396 specimens) ,karyotyping using a standard staining protocol (64 specimens) and cytochrome-b gene sequencing (141 specimens) .Results revealed three forms of Mastomys with different karyotypes in Namibia,Botswana and Angola.These were assigned to species M.coucha (2n = 36,aFN = 60/60) ,M.natalensis (2n = 32,aFN = 57/58) and M.shortridgei (2n = 36,aFN = 51/52) .The mtDNA divergence between the species M.coucha and M.shortridgei was relatively low (1.3%) ,additionally the molecular clock estimated M.shortridgei to be a recent off-shoot of M.coucha (0.71 Mya) .A Model of Drainage Evolution in south-central Africa since the Mesozoic may elucidate the events leading up to the eventual divergence of M.shortridgei from M.coucha.It is estimated that the lake Palaeo-Makgadikgadi,in present day Botswana,covered much of the eastern Kalahari basin.It could be that the peripheral ancestral population of M.shortridgei came in contact with the lake Palaeo-Makgadikgadi and was isolated with the shrinking lake Palaeo-Makgadikgadi during the End-Pleistocene to Early Holocene.Over time M.shortridgei ancestral population became adapted to the local swampy environmental conditions.