Multi-mammate rats (Muridae, Murinae, Mastomys spp.) are Sub-Saharan pest rodents, the systematics of which has long remained confused due to poor morphologic differentiation. Cytotaxonomy and DNA sequencing have provided excellent diagnostic markers for species identification, thus improving the assessment of taxonomic diversity within the genus. In West and Central Africa, three species have been unambiguously identified and are now well documented: Mastomys erythroleucus, Mastomys natalensis and Mastomys huberti. However, recent studies suggest the existence of two other taxa on the basis of morphology, DNA sequencing (Mastomys verheyeni, from Nigeria and Northern Cameroon) or karyotypes (Mastomys cf. kollmannspergeri, from Northern Niger and Chad). In order to clarify the systematic status of the latter taxa, we collected all the available cytotaxonomic as well as cytochrome b sequencing data to which were added results obtained from recently trapped specimens in Northern Cameroon. Our combined molecular and cytogenetic analyses unambiguously confirm the presence of another Mastomys species inhabiting various habitats in Niger, Nigeria, Cameroon, Chad and Sudan. This taxon is clearly differentiated from M. erythroleucus, M. huberti and M. natalensis. Furthermore, the inclusion in our analysis of two M. verheyeni paratypes as well as individuals trapped in close vicinity to the type locality of M. kollmannspergeri supports the assignation of all specimens to M. kollmanspergeri, thus making M. verheyeni its junior synonym. Our study once again highlights the reliability of DNA- and karyotype-based methods to accurately assess specific diversity in African rodents, particularly in the Mastomys genus, which shelters several species of agricultural and epidemiological importance.