Anthropogenic interference with river systems can fragment populations into smaller sub-populations, leading to loss of their genetic diversity necessary to withstand environmental challenges and increasing their susceptibility to evolutionary pressures. This study assessed the genetic diversity and population structure of dam-induced populations of Oreochromis niloticus in the Challawa and Kano Rivers. Four polymorphic microsatellite primers-UNH 146, UNH 172, UNH 185, and UNH 995-were used to amplify extracted DNA of 87 fish samples from four populations located on the two rivers i.e., reservoir (above-dam) and below-dam. Band scores from gel electrophoresis were used for the genetic diversity analysis. The diversity indices, including the number of alleles (Na) per population, effective alleles (Ne), and Shannon’s information index (I), revealed higher values for the reservoir population in the Challawa River compared to its below dam counterpart. Three of the populations displayed heterozygosity deficits (Ho