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Over the past few decades, South American Rivers, including the Paraná River system, have been intensely impacted by dam constructions. To mitigate adverse impacts, fish passages were incorporated into two large dams on the Paraná River (Itaipu and Porto Primavera) to reestablish longitudinal connectivity, which is very important for conserving populations of migratory fish species. The aim of this study was to estimate genetic diversity, population differentiation and gene flow, using microsatellites markers and mitochondrial haplotypes ( d-loop), in populations of Prochilodus lineatus, a long distance migratory species commonly found in Paraná River basin drainages. Samples were obtained from six locations in the Upper and Middle Paraná River: Porto Primavera Reservoir, downstream of the Porto Primavera Reservoir, four tributaries of this reservoir (Pardo, Peixe, Verde and Aguapeí Rivers) and in the Iguaçu River, downstream of the Itaipu dam. High genetic diversity was observed in all samples, as expected for species with large population sizes and high migration rates. However, the influence of other factors, such as secondary contacts, fish passages and restocking were also suggested. In general, evidence of spatial population structuring was not found, even though there were two large dams in the study area. This could also be attributed to the brief period that has elapsed since the dams were built, the influence of migratory and reproductive patterns and the possible contributory effects of the fish passages. Furthermore, the absence of population structuring also corroborates existing information that emphasizes the importance of unimpeded tributaries for conserving migratory fish populations in fragmented watersheds. [ABSTRACT FROM AUTHOR]