The anadromous catfish, Genidens barbus, is a commercial and vulnerable species from South America. The aims of the present study were to assess whether the nursery areas can be discriminated by using microchemical signatures of lapillus otoliths, to assess the accuracy of classifying fish in relation to natal nursery area and to discuss the possibility of existence of homing behaviour. Thus, the otolith-core chemical signatures (barium (Ba):calcium (Ca), lithium (Li):Ca, magnesium (Mg):Ca, manganese (Mn):Ca, strontium (Sr):Ca, and zinc (Zn):Ca ratios) of adult fish were compared among different estuaries (De La Plata River in Argentina, and Patos Lagoon, Paranaguá Bay and Guanabara Bay in Brazil). PERMANOVA analysis showed significant differences in the multi-element signatures of the otolith core among sampling sites for all cohorts (2002, 2003, 2006 and 2007), indicating that the technique has considerable potential for use in future assessments of population connectivity and nursery areas of G. barbus. Via quadratic discriminant function analysis, fish were classified to natal nursery areas with 80–100% cross-validation classification accuracies. These results suggested that a high level of spatial segregation exists in adult catfish life and homing behaviour could not be ruled out on the basis of our data.