The African rodent genus Mastomys contains eight species, some of which are widely distributed pest species and others which have restricted distribution. Most species are recognized only on chromosomal or molecular genetic data. We aimed to clarify the systematic status, geographical distribution, and phylogenetic relationships of three Mastomys species occurring in the Okavango Basin and surrounding arid habitats in southwestern Africa, with a focus on the poorly known and questionably valid taxon, Mastomys shortridgei. We described three genetically, morphologically, and ecologically distinct species of Mastomys and documented their respective karyotypes: Mastomys coucha (2n = 36, aFN = 56); Mastomys natalensis (2n = 32, aFN = 54); and M. shortridgei (2n = 36, aFN = 46). The chromosomal differences between M. coucha and M. shortridgei are probably due to pericentric inversions. The time to the most recent common ancestor of all M. coucha and M. shortridgei was estimated at 0.64 (0.30–1.22) Mya. The three species differed significantly from each other in 11 cranial measurements; a discriminant function allowed us to distinguish co‐occurring M. natalensis from M. shortridgei with 95% accuracy. We propose a model to explain the recent peripatric speciation of M. shortridgei from M. coucha based on drainage evolution in south‐central Africa since the Pleistocene. The conservation status of M. shortridgei needs to be re‐evaluated because of its low population densities, restrictive habitat preference, declining habitat quality, and historical and predicted future declines in area of occupancy.