The phylogeography of the African four-striped mouse, Rhabdomys pumilio, was investigated using complete sequences of the mtDNA cytochrome b gene (1140 bp) and a combination of fluorescence in situ hybridization (FISH) and conventional cytogenetic banding techniques (G- and C-banding). Two cytotypes (2n=46 and 2n=48) were identified by cytogenetic analysis. There is no evidence of diploid number variation within populations, difference in gross chromosome morphology or of subtle interchromosomal rearrangements at levels detected by ZOO-FISH. Analysis of the mtDNA cytochrome b resulted in two major lineages that correspond roughly to the xeric and mesic biotic zones of southern Africa. One mtDNA clade comprises specimens with 2n=48 and the other representatives of two cytotypes (2n=48 and 2n=46). The mean sequence divergence (12%, range 8.3-15.6%) separating the two mtDNA clades is comparable to among-species variation within murid genera suggesting their recognition as distinct species, the prior names for which would be R. dilectus and R. pumilio. Low sequence divergences and the diploid number dichotomy within the mesic lineage support the recognition of two subspecies corresponding to R. d. dilectus (2n=46) and R. d. chakae (2n=48). Our data do not support subspecific delimitation within the nominate, R. pumilio. Molecular dating places cladogenesis of the two putative species at less than five million years, a period characterised by extensive climatic oscillations which are thought to have resulted in habitat fragmentation throughout much of the species range.