The development of a selective breeding program for Nile tilapia (Oreochromis niloticus) in Uganda holds significant potential for enhancing aquaculture productivity. However, the genetic diversity of the species remains inadequately characterized, hampering the establishment of a systematic breeding program, and consequently adversely affecting the production of Nile tilapia. Therefore, the present study explored the genetic diversity and population differentiation among farmed and wild Nile tilapia populations to contribute key insights for designing a systematic breeding program in Uganda. A total of 480 samples from 20 populations, including farmed and wild populations, were collected across Uganda. Microsatellite markers were used for genotyping, and genetic diversity indices, including heterozygosity and FST values, were analyzed. The study employed hierarchical clustering and population structure modeling to assess genetic differentiation. Results indicated that expected heterozygosity (0.50 ± 0.01) was higher than observed heterozygosity (0.42 ± 0.01) in all populations, although the differences were not significantly different. Populations from pond farms exhibited lower FST values (<0.001), suggesting minimal genetic differentiation. The hierarchical clustering identified four major genetic clusters: 1) cage populations (Pal and Busana), 2) Katosi, SON, and Rocks hatchery, 3) Bawe cage fish farm and Tendo hatchery, and 4) populations from ponds and beaches on Lake Victoria. The Tendo and Rocks populations demonstrated higher genetic diversity and differentiation, making them potential candidates for selective breeding efforts. These findings contribute crucial information for the conservation of Nile tilapia genetic resources and support the establishment of a selective breeding program in Uganda.