Abstract Psalidodon fasciatus is a complex of several fish species widely distributed in Brazilian hydrographic systems that share morphological characteristics and show a high taxonomic complexity and genetic variability. Cytogenetic and molecular analyses were carried out in populations from three Brazilian hydrographic basins, aiming to contribute to systematic and biogeographical knowledge of the group. The chromosomal markers verified the occurrence of species-specific characters and indicated the existence of six distinct operational taxonomic units (OTUs): P. fasciatus (São Francisco River basin), Psalidodon sp. 1, Psalidodon sp. 2 and Psalidodon eigenmanniorum (Uruguay River basin); and Psalidodon sp. 3 and Psalidodon sp. 4 (Paraná River basin). The chromosomal markers showed more similarities among species from the Uruguay River and São Francisco River basins. DNA barcoding analyses (assemble species by automatic partitioning, neighbour-joining, maximum likelihood and maximum parsimony) indicated the existence of at least three distinct OTUs. The chromosomal evolution rates were demonstrated to be higher than the molecular evolution rates, reinforcing the importance of using chromosomal markers to delimit OTUs in integrative taxonomy studies. These results suggest that the São Francisco River Basin population (the type locality) should be considered as true P. fasciatus, and the others, until now treated as Psalidodon aff. fasciatus, are cryptic species.