Pacu (Piaractus mesopotamicus) is an important fish species for professional and sport fisheries in the Alto-Paraguay Basin, located in the central western Brazil. In this study the D-loop control region of mtDNA sequence data was used to investigate genetic variability within and among populations of pacu. A total of 99 individuals were collected in the Paraguay River and in four other main tributaries. From these, 27 different haplotypes were observed. The overall nucleotide diversity and haplotype diversity were high in all sampling sites with 0.013±0.001 and 0.840±0.035, respectively. Hierarchical AMOVA analysis showed that pacu populations form a single panmictic population with low Φ ST =−0.0012 (p=0.486) and high gene flow among rivers. Despite high genetic variability, the total capture officially landed in the Upper-Paraguay River Basin has been decreasing steadily during the last years with indications of overexploitation. Taking into account the heterogeneity of habitats, continuous anthropogenic disturbances and lack of genetic structure, it appears that fish from these sampling sites function as a metapopulation, a framework that may be applied to long term fishery management of pacu in the Panatanal.