The freshwater fish Piaractus brachypomus is an economically important for human consumption both in commercial fisheries and aquaculture in all South American countries where it occurs. In recent years the species has decreased in abundance due to heavy fishing pressure. The species occurs in the Amazon and Orinoco basins, but lack of meristic differences between fishes from the 2 basins, and extensive migration associated with reproduction, have resulted in P. brachypomus being considered a single panmictic species. Analysis of 7 nuclear microsatellites, mitochondrial DNA sequences (D-loop and COI), and body shape variables demonstrated that each river basin is populated by a distinct evolutionarily significant unit (ESU); the 2 groups had an average COI divergence of 3.5% and differed in body depth and relative head length. Historical connection between the 2 basins most probably occurred via the Rupununi portal rather than via the Casiquiare canal. The 2 ESUs will require independent fishery management, and translocation of fisheries stocks between basins should be avoided to prevent loss of local adaptations or extinction associated with outbreeding depression. Introductions of fishes from the Orinoco basin into the Putumayo River basin, an Amazon basin drainage, and evidence of hybridization between the 2 ESUs have already been detected.