The study of species in their native geographic ranges is key to understanding how human activity has influenced spatial fragmentation or species homogenization. The Argentinian silverside Odontesthes bonariensis, of interest for aquiculture and sport fishing, is a relevant subject of study. The species has been introduced in a number of countries and re-introduced in some areas of Argentina with unknown effects. The objectives of this study were to determine the population structure, genetic diversity (GD) and effective population sizes (Ne) of O. bonariensis in Argentina. Six microsatellite loci were amplified in individuals collected from four water bodies affected by commercial and sport fishing: Cabra Corral Reservoir (CC), Chascomús Lake (CH), Chasicó Lake (LCH) and the Río de la Plata (RLP). Three genetic groups were detected: one in CC, one in RLP and the last inhabiting CH and LCH. Interestingly, CH and LCH are located 768 km apart, but showed no difference in allele frequencies; suggesting the introduction of individuals from CH into LCH. The largest allele richness, GD and Ne were found in RLP indicating that the largest population of O. bonariensis may be found in this area. Current Ne were lower than historical Ne in all areas, suggesting a change in the GD over time. This study provides information on the genetic structure and genetic diversity of O. bonariensis across its native distribution and over time, demonstrating the first evidence of a possible genetic homogenization in this species probably linked to human activities.