The two common southern African mice species (Mastomys coucha and M. natalensis) are morphologically almost identical, making field identification impossible at present. Specimens from two localities were collected and tissue and blood samples taken. The habitat type of each locality was studied, and a distribution map compiled. A definite correlation between biome-type and species range was found to be present. Three isozyme markers were identified: glucose phosphate isomerase in liver, and two general (non-specific) protein coding loci in muscle. In addition, we also identified species characteristic haemoglobin components in both species. This is the first study to report genetic variation within, and differentiation between these species. Our results are of medical importance because Mastomys coucha carries bubonic plague and M. natalensis carries Lassa Fever.