Fish stocking programs have been implemented to mitigate the blockage of original riverbeds by the construction of hydropower dams, which affects the natural migration of fish populations. However, this method raises concerns regarding the genetic rescue of the original populations of migratory fish species. We present the first study to investigate the genetic diversity, population structure, and gene flow (migration) of the Neotropical migratory fish Prochilodus costatus in the Três Marias dam in São Francisco River basin, Brazil, using high-throughput genotyping and population genetics approaches. In total, 1,017 specimens were sampled from 12 sites during 4 hydrological years and from broodfish used in a fish stocking program, and genotyped with 8 microsatellite loci. The samples showed low genetic variability, with inbreeding and the presence of only four genetic pools; three pools were observed throughout the study region and the fourth was exclusive to one area in the Paraopeba River. Additionally, we identified high unidirectional gene flow between regions, and a preferred route between the Pará river and the upper portion of the São Francisco River. The fish stocking program succeeded in transposing the genetic pools from downstream to upstream of the Três Marias dam, but, regrettably, promoted genetic homogenization in the upper São Francisco River basin. Moreover, our data show the fragility of this species at the genetic level, and through these strategic methods of monitoring that could be as model for migratory fish populations that are consumed by humans to develop conservation and management measures.