Zimbabwe’s smallholder land-based livelihoods are dominated by the three local Sanga cattle breeds, namely Mashona, Tuli, and Nkone. A study was carried out to determine genetic diversity and differentiation among conservation populations of these breeds using 16 bovine-specific microsatellite markers. These markers included BM1818, BM1824, BM2113, CSRM60, CSSM66, ETH10, ETH225, ETH3, ILST006, INRA23, RM067, SPS115, TGLA122, TGLA126, TGLA227, and TGLA53. All marker loci contributed to breed differentiation based on the infinitesimal model (FST), with the most powerful markers being CSSM66 (25%), ETH225 (20.6%), and TGLA122 (13.8%) and the least powerful being RM067 (0.7%), BM1824 (1.0%), and ETH10 (1.1%). Three marker loci (BM1824, ETH225, and ETH3) revealed significant deviations from Hardy-Weinberg equilibrium (HWE) proportions. A total of 119 alleles were observed, ranging from 4 to 11 and averaging 7.4 alleles per locus. Thirty-four of these alleles were unique to specific breeds. Mean (Na) and effective (Ne) numbers of alleles were 5.167 ± 0.302 and 3.462 ± 0.163 alleles per locus, respectively, with no significant differences between breeds. Observed heterozygosity (HO) (0.73) was higher than expected heterozygosity (HE) (0.71), revealing that breeds were in HWE. Global F-statistics FIT, FST, and FIS gave mean values of 0.059, 0.084 and -0.028, respectively. Overall breed genetic differentiation was moderate (FST = 8.4%) and significant (P <0.001). Multivariate analyses separated the three breeds completely, and revealed that most of the genetic variation was within breed (92%). The analyses revealed that a significant amount of variation is maintained in these breeds and that they are distinct genetic entities that may be considered for utilization or conservation.