Podocnemis sextuberculata (Pleurodira: Podocnemididae) is widely distributed throughout the Amazon drainage basin in Brazil, Colombia, and Peru. Telemetry and previous molecular data suggest that P. sextuberculata lacks population structure in the central Amazon basin of Brazil. Generalization of these results, however, requires much-broader sampling across a range of habitats of this broadly distributed species. For this reason, we tested the hypothesis of panmixia in P. sextuberculata, analyzing a total of 319 specimens sequenced for the mitochondrial control region. Our sampling included localities from 16 areas in the Amazon basin from rivers characteristic of the Amazon basin (whitewater), clearwater rivers of the Guiana shield (Branco, Trombetas, and Nhamundá rivers), and the Brazilian shield (Xingu River). The hypothesis of panmixia was rejected because the results of analysis of molecular variance, pairwise ФST, and Bayesian analysis of population structure indicated population structure in the group of individuals from the locality of Xingu which was not correlated to a pattern of isolation by distance. We suggest that the populations of P. sextuberculata of the Brazilian Amazon basin are composed of 2 management units, one represented by populations restricted to the Xingu River and the other represented by all other populations. The population of the Xingu should be viewed with attention and concern, especially considering the direct and indirect impacts of damming the Xingu River.