In order to evaluate the contribution of geological, environmental, and climatic changes to the spatial distribution of genetic variation of Mastomys natalensis, we analysed cytochrome b sequences from the whole distribution area of the species to infer its phylogeographic structure and historical demography. Six well-supported phylogroups, differentiated during the Pleistocene, were evidenced. No significant correlation between genetic and geographic distances was found at the continental scale, and the geographic distributions of the observed phylogroups have resulted from extensive periods of isolation caused by the presence of putative geographic and ecological barriers. The diversification events were probably influenced by habitat contraction/expansion cycles that may have complemented topographic barriers to induce genetic drift and lineage sorting. According to our results, we propose a scenario where climate-driven processes may have played a primary role in the differentiation among phylogroups.