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The subfamily Serrasalminae (Characidae, Teleostei) is an endemic Neotropical group of fishes distributed along the Amazon, Paraná‐Paraguay and Orinoco Basins. The pacu (Piaractus mesopotamicus) is found in all major rivers that comprise the Pantanal of Mato Grosso. In order to investigate population genetic structure within the group, an enriched microsatellite library was constructed. Eight polymorphic were loci screened, with an average of 6.5 alleles per locus. Five loci exhibited greater than 60% heterozygosity. Additionally, a high level of cross‐species amplification was obtained.