The La Plata River basin, the second largest drainage basin in South America, supports important fisheries, in which the sábalo Prochilodus lineatus is the leading species measured as extracted volumes. To characterize the population structure of the sábalo in the middle and lower regions of the La Plata River basin, cytochrome b DNA sequences of 121 samples collected in La Plata, Paraná and Uruguay rivers were used. Results suggest the presence of at least two populations: one that we called “southern population” uses the La Plata River and the lower sections of the Paraná and Uruguay rivers and another population (called here “northern population”) in the middle section of the Uruguay and Paraná rivers. The southern population would have a larger effective population size and coalescence-based analyses reveal an asymmetric gene flow, mainly from north to south. A striking result is that samples from the middle sections of the Paraná and Uruguay rivers are more alike to each other than they are to the other samples of their respective rivers. This similarity could be explained by historical reasons, probably as a consequence of the proximity that both rivers had until 100,000 years ago. The presence of different management units being exploited by the continental fisheries of three countries demands coordination at a regional level in order to maintain a sustainable fishery.