Landscape characteristics can influence gene flow depending on the species ability to disperse. This imposes different levels of resistance to movement, determining the genetic structure and diversity of populations. Waterfalls and rapids in the Amazon basin have been suggested as contributing factors to the diversification and genetic structure of freshwater species, including turtles. Thus, along some main rivers of the Brazilian Amazon: Juruá, Madeira, Amazonas, Trombetas, Tapajós, Xingu, Tocantins, and Araguaia, we evaluated the spatial distribution of genetic diversity and characterized the population structure of Podocnemis unifilis, a widely distributed and endangered Amazonian turtle, using the mitochondrial DNA control region. We tested for isolation by geographic distance and by resistance models through an integrative approach using genetic, geographic, and ecological data, comparing these matrices with genetic distance. We found heterogeneous levels of genetic diversity and no spatial patterns. Results supported the isolation by resistance hypothesis, where the movement of individuals was influenced by freshwater, vegetational, climatological, and geomorphological variables, explaining the genetic distance of P. unifilis. Rapids and waterfalls impose greater resistance although they are barriers with different levels of permeability to the P. unifilis gene flow. Thus, the idiosyncrasies of each sub‐basin must be considered in the interpretation of the patterns found in assessing phylogeographic history.Abstract in Portuguese is available with online material.