Recent studies have described the presence of several mitochondrial lineages within Rhabdomys, which was previously considered to be a monotypic genus. The exact distributional limits of the species and subspecies and their contact zones are unclear. In this study we demonstrate that two monophyletic Rhabdomys dilectus mitochondrial lineages are present at two northern Gauteng province sampling sites in South Africa. Cytochrome b gene sequences, 896 nucleotides in length, generated for 36 Rhabdomys samples identified 10 unique haplotypes corresponding to eight R. dilectus dilectus haplotypes (from 32 individuals) and two R. d. chakae haplotypes (from four individuals). The present study provides the first empirical evidence for a contact zone for two R. dilectus conspecific mitochondrial lineages and contributes to the refinement of Rhabdomys distributional maps in southern Africa.