We analysed 131 common impala (Aepyceros melampus melampus) samples from two provinces in South Africa (Limpopo and KwaZulu-Natal) that are separated by the Drakensberg Mountain Range using sequences of the mitochondrial control region and seven polymorphic nuclear microsatellite loci. In line with earlier studies on bovid species, we found very high values of genetic diversity, particularly at the mtDNA locus with an overall nucleotide diversity of 3.6% and an overall haplotype diversity of 0.98. All statistical approaches confirmed a significant population differentiation between Limpopo and KwaZulu-Natal, suggesting that areas of unsuitable habitat caused by the presence of the Drakensberg Range and the Indian Ocean coastal belt act as a barrier to gene flow. Only few individuals with signs of admixed origin were indicative of translocations or rare migration between the two provinces. Combination of our mtDNA data set with those of previous studies on impala from south-western, southern and eastern Africa revealed the highest diversity in South Africa. This is in line with the hypothesis of a southern glacial refuge from which various African ungulate species spread northeast during the Holocene, although in the case of impala further analyses based on larger data sets will be necessary to definitively settle this question.