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ABSTRACT DNA barcoding is a widely utilized molecular-based identification of species and taxonomic resolutions. Until recently, Rhamdia voulezi and Rhamdia branneri were considered species synonyms of Rhamdia quelen; however, morphological and cytogenetic analyses have suggested the validity of distinct species. Due to the absence of molecular taxonomy of R. voulezi and R. branneri, the objective of this study was to test its validity through traditional DNA barcoding and the GMYC (General Mixed Yule Coalescent) COI-based analyses in 19 specimens from the Iguaçu River Basin. In both methodologies, three MOTUs (Molecular Operational Taxonomic Units) were identified based on the estimated optimum threshold (OT = 0.77). The average inter-MOTU distance (NJ, K2P) between R. branneri and R. voulezi was 1.4%, and 0% intra-MOTU distance in both species. The two species identified as R. branneri and R. voulezi showed correspondence with taxonomic and morphological identifications. With regard to R. quelen, the average intra-MOTU distance was greater than OT (2.7%), indicating that this species can be formed by different MOTUs. We suggest that molecular and taxonomic studies should be employed concurrently in R. quelen, to prevent contamination of wild species by hybridizations

DNA barcoding confirms the occurrence of Rhamdia branneri and Rhamdia voulezi (Siluriformes: Heptapteridae) in the Iguaçu River Basin