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In the neotropics, potamodromous fish species are among the most severely impacted by dams, which can lead to blocking of the migratory routes, elimination of the breeding areas, and population isolation in this group. However, growing evidence has indicated that free-flowing tributaries along fragmented watersheds provide important contributions to the reproductive migration and recruitment of migratory fish. If this is really occurring, we could expect low or undetectable population differences in different reservoirs and respective unimpeded tributaries along watersheds fragmented by dams. Aiming to test this hypothesis, we assessed the genetic diversity and population structure of Pimelodus maculatus, a migratory catfish species, in seven reservoirs and nine tributaries along the Paranapanema River basin, Paraguay-Paraná-Plata system, South America. As predicted in our initial hypothesis, after analyzing nine microsatellite loci and a 406-bp mitochondrial DNA sequence (D-Loop) in 640 individuals, we found high nuclear genetic diversity and non-significant genetic substructuring among each reservoir and its tributaries, contrasting with the results between samples separated by dams. Our findings reinforce the idea that free‐flowing tributary rivers are contributing to maintenance of wider population distributions, larger effective population sizes, and satisfactory genetic diversity levels. [ABSTRACT FROM AUTHOR]