Stock composition studies using otolith fingerprints have scarcely been developed in freshwater systems. In this context, the La Plata Basin is the second largest fluvialmarine system of South America, where Prochilodus lineatus is the most important fishery resource. Despite the basin importance, estimation of mixed stock composition has not been explored yet. In this paper, the contribution of the two main nursery areas to two fishing stocks (Uruguay and Paraná rivers) of P. lineatus was evaluated using otolith's core microchemistry, considering two groups: subadult (2 years) and adult (4 years). Estimates were made using two maximum likelihood methods. Chemical composition of young-of-year fish caught in nursery areas in 2010 was used as baseline of the models, whereas chemical composition of the core of subadult and adult otoliths was used as sample of unknown origin. Results suggest that the subadult stock from Paraná was not mixed (contribution~100%), whereas the stock from Uruguay had a contribution from the Paraná nursery (1.5 ± 1.2-17.9 ± 3.96%). For the adults, the degree of mixing increased and the contribution from both nursery areas to the Paraná and Uruguay stocks varied between 14.8 ± 4.18% and 85.2 ± 4.18%, respectively. The potential application of otolith fingerprints and maximum likelihood mixture models is here highlighted for determining the relative importance of recruitment sources of fish in the La Plata Basin. Because the contributions of the different nursery areas shared among several nations in turn affect the composition of internationally shared stocks, comprehensive management agreements at the basin level are necessary.