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Habitat fragmentation caused by hydroelectric dams has depleted fish populations worldwide. Restocking actions are usually adopted to recover those populations, but hatchery management protocols rarely guarantee the maintenance of genetic diversity and a balanced contribution among captive breeders each generation. Here, a set of 10 microsatellite markers was used to assess the genetic diversity (average allelic richness AR?=?10.87 and expected heterozygosity HE?=?0.742, respectively) and structuring of Megaleporinus obtusidens, a migratory freshwater fish inhabiting over 2500 km of the São Francisco River in Brazil. Three main genetic clusters were identified in this species across the river basin that could be related to the sharply different climatic and hydrologic regimes from the Upper to the Lower course. A significant reduction (> 50%) in genetic diversity was observed in the broodstock when compared to their wild conspecifics, especially in the allelic richness. The information here presented will aid for management of genetic resources of this species in the São Francisco River taking as reference the genetic clusters identified. Furthermore, the results indicated that restocking is not necessary unless signals of population depletion occurs and, if so, hatchery reproductive protocols should rely on artificial fertilization rather than mass spawning.

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