Prochilodus lineatus is a species with high economic value that easily adapts to captive breeding. Hence it became a great interest for the development of fish breeding programs. Regular monitoring of genetic variation in natural fish stocks, especially in species of interest in conservation programs, is necessary to avoid a decline of genetic variability, which is of utmost importance for species conservation. In this sense, the objective of this study was to estimate the different genetic structures of P. lineatus and features of phylogeographic structures using mtDNA sequences of ATP synthase subunits 6 and 8 gene. The Neighbor-Joining analysis presented all kinds of population grouped in one clade. We detected low genetic distance values among the population (0, 5%), and 27, 4% for comparisons with the outer group. The low genetic distance and low AMOVA FST values suggest that, the population of the Mato Grosso Pantanal is panmictic.