We used the DNA barcoding technique (COI and CytB markers) combined with GMYC analysis to characterize the genetics of the widely distributed Neotropical fish species Knodus moenkhausii from three different isolated hydrographic basins. Despite the fact that most of the Neotropical hydrographic basins have been isolated for millions of years, species could be shared between basins due to natural events (stream capture) or anthropogenic activities. Recent surveys, however, have shown that many widely distributed species are actually species complexes divided into previously unrecognized cryptic species. In this study, we tested the hypothesis that K. moenkhausii from three hydrographic basins represent a single panmictic species and discuss the most likely explanation of its present geographical distribution. The GMYC analysis revealed that all specimens of K. moenkhausii represent a single species: the intra- and intergroup minimum K2P genetic distances for both genes were zero and haplotypes were shared among the three hydrographic basins. This suggests there has been recent interchange of K. moenkhausii throughout the three hydrographic basins. It is likely that this is due to recent human activities, either the transposition of natural barriers or intentional introduction or accidental escape due to the ornamental fish trade.