M.Sc. / Two species of multimammate mice occur in South Africa, namely Mastomys natalensis and M. coucha. These species, both reservoirs for different and equally important disease pathogens, are morphologically identical with variations described in chromosome number, sperm morphology, pheromones, ultrasound and allozyme and isozyme markers. Their identical macro morphologies also raise problems where habitat and distribution is concerned. The need to identify various disease threats based on the carrier organism makes their identification important because they occur sympatrically in many areas of South Africa. Both species were expected to occur within the Roan Camp, Kruger National Park, South Africa and therefore toe samples were obtained from D. MacFadyen who was able to capture the Mastomys genus during his biodiversity survey of the area. He was unable to assign species designations to the captured individuals based on external morphology, but identification would allow exploration into the habitat preferences of each species when compared to the vegetation data gathered by MacFadyen using cross tabulation methods. DNA was extracted from 90 toe samples obtained during August 2003 (30 samples) and 2004 (60 samples) and the cytochrome-b region was amplified using the polymerase chain reaction (PCR). Amplified PCR products were cut using the restriction enzyme BsmAI to produce restriction fragment length polymorphism (RFLP) profiles from which the two species were identified. Once identified, the species were associated to vegetation data received from MacFadyen and samples were selected for further analysis using 5 microsatellite loci. Mastomys natalensis was found in very low numbers, as expected, based on the average rainfall and altitude of the sample area, which is preferred by M. coucha. Further insight was gained into the preferences of each species concerning habitat and their respective status as pioneer species in habitat recolonisation. Genetic differences were obtained within the two species with 100% and 80% polymorphic loci for M. coucha and M. natalensis respectively. Allele classes at most of the loci deviated from Hardy-Weinberg equilibrium, probably due to small sample sizes, null alleles and/or heterozygote deficiencies with average heterozygosities of 0.754 for M. coucha and 0.526 for M. natalensis. The genetic distance between the two species was 0.465 and an Rst value of 0.503 indicated a significant differentiation between the two species. Fixed allele differences between the species were not obtained, but private alleles were found at four loci in M. coucha and one locus in M. natalensis. This study therefore contributes to the global information on species identification, genetic variation and ecology of the two cryptic Mastomys species; the three pillars on which the Convention of Biological Diversity (of which South Africa is a signatory) rests upon.