Genetic variability in three species of Pimelodidae catfish (Iheringichthys labrosus, Pimelodus maculatus and Pimelodus cf. absconditus), collected at four sites in the Tibagi river-basin: Sertaneja, Londrina (lower Tibagi), Sapopema (middle Tibagi) and Tibagi (upper Tibagi) was analysed using RAPD and isoenzymatic techniques. Iheringichthys labrosus had the greatest genetic variability showing a proportion of mean polymorphic loci (P¯m) of 53.7%. The dendrogram showed that the individuals from the locations were clustered together, supporting the hypothesis that I. labrosus has a sedentary or localized geographical distribution. Pimelodus maculatus is the only species studied that is considered to be highly migratory. The P¯m obtained for this species was 30.2%. Their genetic dendrogram showed that the individuals were not clustered, based on the place of collection. P. cf. absconditus had a P¯m value of 47.4%, and the external morphology of this species is similar to that of I. labrosus, which hindered differentiation of individuals from the two species. RAPD and isoenzyme analysis, however, provided differential patterns for P. cf. absconditus from I. labrosus.