Pisciculture has been an important part of the economy in many regions of Brazil, and the tambaqui (Colossoma macropomum) stands out as one of the country’s most commercialized native freshwater fish species. Loss of genetic variability can affect characteristics such as reproduction and growth rates, as well as disease resistance and is of great concern in this field. Molecular markers such as mitochondrial DNA genes have been increasingly used to understand genetic variability in species of economic importance. This study aimed to characterize the genetic variability of tambaqui populations reared in two fish farms (Itiúba, AL and Betume, SE) of the lower São Francisco River by analyzing the control region and ATPase genes of mitochondrial DNA. Sequencing of samples from progenies and breeding individuals identified 42 haplotypes (32 unique), of which four haplotypes were shared between the two fish farms as a result of a founder effect, because individuals from Itiúba formed the stock of the Betume fish farming Station. Haplotype diversity was high in both locations. Analysis of Molecular Variance (AMOVA) revealed greater genetic variation within populations (96.76%) than between them (3.24%) and the FST value (0.03) indicated low genetic differentiation between the two populations. The observed high genetic variability can be explained by the number of breeders in the two fish farms (200 in Itiúba and 1400 in Betume) and the breeding management system, which seems to be effective in maintaining stock variability.