The Namaqua Afrikaner is an endangered sheep breed indigenous to South Africa, primarily used in smallholder farming systems. Genetic characterization is essential for the breed’s conservation and utilization. In this study, a genetic characterization was performed on 144 Namaqua Afrikaner sheep kept at the Karakul Experimental Station (KES), Carnarvon Experimental Station (CES), and a private farm Welgeluk (WGK) using 22 microsatellite markers. The mean number of alleles observed was low (3.7 for KES, 3.9 for CES, and 4.2 for WGK). Expected heterozygosity values across loci ranged between 46 % for WGK, 48 % for KES, and 55 % for CES, indicating low to moderate genetic variation. The analysis of molecular variance revealed that 89.5 % of the genetic variation was due to differences within populations. The population structure confirmed the differentiation of three clusters with high relationships between the CES and WGK populations. In the population structure comparison with Pedi and South African Mutton Merino sheep, limited hybridization between the Namaqua Afrikaner sheep and both of these breeds was observed. The results of this study will serve as a reference for genetic management and conservation of Namaqua Afrikaner sheep.