Curimatã-pioa (Prochilodus costatus) and curimatã-pacu (Prochilodus argenteus) are migratory fish species endemic to the São Francisco River Basin in Brazil. Both species play important roles in local fisheries and ecology in the Paraopeba River. A dam was recently constructed on this river and to help in the development and conservation programs, we characterized the genetic variation of both species before dam construction. Complex hypervariable repeats microsatellite was used to asses genetic variation for both species within and between the five collection sites in order to detect population substructuring. Nucleotide substitutions and insertion/deletion polymorphisms (indels) resulted in 35 P. costatus haplotypes (sample size = 89) and 22 P. argenteus haplotypes (sample size = 32). Significant genetic diversity and population differentiation was detected between five sampling sites for both species. Therefore, each of the five sites should be regarded as a group comprising significant genetic differences in species conservation and maintenance plans. Comparing these results to genetic diversity measures after dam construction will be critical for future management in this region.