INTRODUCTION: Southern Africa has been inhabited by hunter-gatherers for at least 20,000 years and has received diverse immigration flows in the last 2000 years. The original inhabitants have interacted with the pastoralist migrants from Eastern Africa (∼2000 ybp), followed by the southern Bantu migration arriving some 1000 ybp, and more recently with the European and Asian settlers after the 17th century. Many of the original Khoekhoe and San inhabitants have either become extinct or have disappeared through admixture in South Africa (SA), in a sex-biased manner involving KhoeSan women. METHODS: In this study, we generated mitochondrial DNA (mtDNA) control region (CR) sequences for 247 South African individuals. The sampling effort was concentrated in regions and populations with historical links to the KhoeSan population groups: admixed (Coloured, Griqua), Nama (Khoekhoe) and Bantu in three provinces. Here we evaluate the composition and extent of connectivity between population groups and regions, and to assess the distribution of haplotypes for the practical application of mtDNA CR data in forensic identifications. RESULTS: The analysis of the newly generated sequences revealed 142 distinct haplotypes, of which 122 were unique. Haplogroup L0 was predominant (overall 71.7%). A high-frequency L0d2a haplotype dominated the pool of the admixed groups with 10%-12.5% incidence overall or per region. Comparative analysis with 545 extant mtDNA CR sequences from South African KhoeSan and admixed descendants revealed extensive population structure and high within-group haplotype sharing. CONCLUSION: The observed population and regional variations, combined with the prevalence of high-frequency haplotypes, align with patterns of matrilocality. These findings highlight the limitations of using mtDNA control region analysis for forensic applications in South Africa.