The systematics of the genus Mastomys in southern Africa is unstable and has experienced many changes over the years. Small mammals of the genus Mastomys are important reservoir hosts of many diseases affecting humans and they are significant agricultural pests. However, it is not clear if the different species affect humans in a similar way. Therefore, knowledge about taxonomic status and the distribution range of the members of the genus Mastomys in southern Africa is of great importance. This study aims to summarise the patterns of morphological, cytogenetic and genetic variation of genus Mastomys across the south-west arid region of southern Africa, as well as clarifying the taxonomic status, identification keys and distribution limits in this region. A total of 352 specimens were trapped in these countries. The methodological approach included skull morphometrics- both traditional and geometric- (396 specimens), karyotyping using a standard staining protocol (64 specimens) and cytochrome-b gene sequencing (141 specimens).