Tilapia is native to Africa, and one of the most cultivated fish in the world. The goal of this research was to evaluate the genetic diversity of tilapia Oreochromis mossambicus from the Limpopo, Incomati, Umbeluzi and Sabié rivers in Mozambique. Microsatellite markers were used to assess the genetic structure and to compare the genetic variability of wild populations of O. mossambicus. DNA samples from 200 specimens were analyzed. All five loci (UNH104, UNH129, UNH142, UNH222 and UNH231) used in this study were polymorphic, with observed heterozygosity ranging from 0.940 to 1.000 and the allelic richness average (Ar) ranging from 8.937 to 15.751. All of the stocks exhibited a remarkably significant excess of heterozygosity relative to the Hardy-Weinberg Equilibrium. Evidence of a genetic bottleneck was found in the four populations evaluated herein. The genetic structure of the population was investigated using the analogues FST and DEST. The most genetic variability occurred within populations. Differentiation among populations ranged from low to moderate levels. No significant correlation was found between geographic and genetic distances. Implications of these findings for management and conservation of O. mossambicus stocks are discussed.