In contrast to the other Podocnemis species, Podocnemis erythrocephala (Pleurodira: Podocnemididae) is restricted to blackwater and clearwater rivers and lakes, including the Negro River basin, and several other tributaries of the eastern Amazon River basin. In order to test the effects of geographic barriers on genetic connectivity of P. erythrocephala, 10 localities sampled throughout the Brazilian Amazon basin were studied. Two hundred and forty-six specimens were sequenced for the mitochondrial control region. Analysis of molecular variance confirmed the existence of population subdivision (ΦST = 0.28, p < 0.001). In pairwise comparisons, the values of the fixation index ΦST were significant in almost all comparisons involving the São Gabriel da Cachoeira (Negro River), Jaú National Park (Jaú River), and Barreirinha (Andirá River), indicating that these populations are genetically differentiated. Genetic differentiation is most likely explained by the presence of rapids and waterfalls separating the São Gabriel da Cachoeira (Negro River) and Jaú National Park (Jaú River) populations from the remaining populations. Populations from Barreirinha (Andirá River) were also genetically differentiated, providing support for the hypothesis that the Amazon River is a geographic barrier for this species. Although distributed over a large geographic area, the remaining 7 localities were not differentiated from one another (p > 0.005), suggesting that these localities are part of a panmictic population distributed throughout the central Amazon basin. We recommend that the 4 structured populations be treated as separate management units.