MSCAGR (Animal Science) / Department of Animal Science / The Nguni is a transboundary indigenous Southern African cattle breed. The breed has distinct populations that are adapted to the different ecological zones of Southern Africa. Previous work on characterising the Nguni has been limited to within-country studies. Thus, the aim of the current study was to genetically characterise South African (SA) Nguni, Mozambican Nguni (Landim) and Swazi Nguni populations across Southern African region using a panel of 25 microsatellite markers, recommended by FAO and ISAG for genetic diversity studies. Genotypic data were generated from 90 unrelated autosomal DNA samples of the three cattle populations (SA Nguni n=30, Mozambican Nguni (Landim) n=30 and Swazi Nguni n=30) collected from government research stations and stud herds. Five South African beef cattle breeds’ DNA profiles were obtained from the ARC-DNA database and used as reference populations. A majority of the microsatellite markers were highly polymorphic across the studied populations. High genetic diversity was detected and expected heterozygosity varied from 71% (Landim) to 75% (SA Nguni) with a higher mean number of alleles (MNA) in the SA Nguni (7.52±0.42) compared to the Swazi Nguni (6.92±0.40) and Landim (7.16±0.43) populations. Observed heterozygosity (Ho) (0.597±0.046) compared to expected heterozygosity (He) (0.719±0.022) was lowest for the Swazi Nguni, confirming a relatively high level of inbreeding (FIS=0.158) in that population. An analysis of molecular variance (AMOVA) revealed that 9.61% of the total variation occurred among populations, while 90.39% occurred within populations. Short genetic distance (29.9%) was observed between Landim and Swazi Nguni, with the SA Nguni (>50%) being the most genetically distant population. The distant relationship between SA Nguni and the other two Nguni cattle populations was further confirmed by neighbor-joining (NJ) tree, Principal Coordinates Analyses (PCoA) and Factorial Corresponding Analysis (FCA). The structure of the three Nguni cattle populations clustered independently, despite some evidence of admixture. Additionally, genetic differentiation and population structure within four Mozambican indigenous cattle populations were investigated using the same panel of microsatellite markers. The analysis of unrelated autosomal DNA was performed on 120 animals (Angone n=30, Bovine de Tete n=30, Landim n=30 and Namaacha Nguni n=30), which presented sufficient genetic diversity across all populations. Estimates of mean number of alleles, observed and expected heterozygosities were 6.920±0.20, 0.68±0.02 and 0.71±0.01, respectively. Genetic differentiation among the populations accounted for 8.02% of total genetic variability. Negative (-0.025±0.029) to low positive (0.073±0.050) levels of inbreeding were observed within the four populations. The genetic distance, NJ tree, PCoA and FCA revealed a close relationship between Bovine de Tete and Landim as opposed to Angone and Namaacha Nguni. STRUCTURE analysis assigned the four Mozambican populations independently; however Bovine de Tete and Landim showed relatively higher levels of admixture with each other than Angone and Namaacha Nguni. It can be concluded that SA Nguni, Landim and Swazi Nguni populations accomplish high genetic diversity and they are genetically distant; however, the two latter populations are closely related. These results present useful information / NRF