Selection has created a range of diverse breeds, important for genetic studies. To date studies have been conducted to evaluate genetic diversity in cattle (Rege 1999; Toro et al., 2010), but this has not been undertaken in Zambia. Our study used genetic information from 32 microsatellites in 72 Zambian Bos indicus breeds (Tonga, Tonga and Barotse) to assess genetic diversity and population structure. Results demonstrate that Angoni breed exhibited  slightly excess average observed heterozygosity (1.0%), while Barotse and Tonga breeds exhibited slight deficit observed heterozygosity of (0.09% and 3.6%) respectively. Global heterozygosity deficit across populations (Fit=4.2%), significantly different from zero (p<0.001), because of observed inbreeding within breeds (Fis=1.0%).These breeds were slightly genetic differentiation (Fst= 3.2%), significantly different from zero (p<0.001). High gene flow (Nm=11.3%) was evident between populations. Although, these breeds didn’t exhibit a high and unique breed’s purity, cattle exhibited higher level of genetic diversity within breeds than between breeds, despite the evidence of close gene flow between the three populations going by Bayesian cluster at K=2. This suggests evidence of existing divergent and multi-loci genetic admixtures between and within breeds. This uniqueness of population clustering offers valuable information on available gene pool for utilisation, improvement and conservation.