Red‐bellied piranhas ( Pygocentrus nattereri) are widely caught with different intensities throughout the region of Solimões‐Amazonas River by local fishermen. Thus, the management of this resource is performed in the absence of any information on its genetic stock. P. nattereri is a voracious predator and widely distributed in the Neotropical region, and it is found in other regions of American continent. However, information about genetic variability and structure of wild populations of red‐bellied piranha is unavailable. Here, we describe the levels of genetic diversity and genetic structure of red‐bellied piranha populations collected at different locations of Solimões‐Amazonas River system. We collected 234 red‐bellied piranhas and analyzed throughout eight microsatellite markers. We identified high genetic diversity within populations, although the populations of lakes ANA, ARA, and MAR have shown some decrease in their genetic variability, indicating overfishing at these communities. Was identified the existence of two biological populations when the analysis was taken altogether at the lakes of Solimões‐Amazonas River system, with significant genetic differentiation between them. The red‐bellied piranha populations presented limited gene flow between two groups of populations, which were explained by geographical distance between these lakes. However, high level of gene flow was observed between the lakes within of the biological populations. We have identified high divergence between the Catalão subpopulation and all other subpopulations. We suggest the creation of sustainable reserve for lakes near the city of Manaus to better manage and protect this species, whose populations suffer from both extractive and sport fishing.