In this study, we compare the level and distribution of genetic variation between South African conserved and village chicken populations using microsatellite markers. In addition, diversity in South African chickens was compared to that of a reference data set consisting of other African and purebred commercial lines. Three chicken populations Venda, Ovambo and Eastern Cape and four conserved flocks of the Venda, Ovambo, Naked Neck and Potchefstroom Koekoek from the Poultry Breeding Resource Unit of the Agricultural Research Council were genotyped at 29 autosomal microsatellite loci. All markers were polymorphic. Village chicken populations were more diverse than conservation flocks. structure software was used to cluster individuals to a predefined number of 2 ≤ K ≤ 6 clusters. The most probable clustering was found at K = 5 (95% identical runs). At this level of differentiation, the four conservation flocks separated as four independent clusters, while the three village chicken populations together formed another cluster. Thus, cluster analysis indicated a clear subdivision of each of the conservation flocks that were different from the three village chicken populations. The contribution of each South African chicken populations to the total diversity of the chickens studied was determined by calculating the optimal core set contributions based on Marker estimated kinship. Safe set analysis was carried out using bootstrapped kinship values calculated to relate the added genetic diversity of seven South African chicken populations to a set of reference populations consisting of other African and purebred commercial broiler and layer chickens. In both core set and the safe set analyses, village chicken populations scored slightly higher to the reference set compared to conservation flocks. Overall, the present study demonstrated that the conservation flocks of South African chickens displayed considerable genetic variability that is different from that of the assumed founder populations (village chickens).