Pimelodidae family is one of the most diverse and widely distributed fish groups in South America. Phylogenetic analysis in the family have recently indicated the existence of two main clades: "sorubiminae" and the OCP clade, including Pimelodus ornatus, "calophysines" and "pimelodines." The aim of this study was to investigate the karyotype of three Amazonian Pimelodidae species: Calophysus macropterus, Propimelodus eigenmanni, and Exallodontus aguanai associating them to the literature, seeking to reconstruct probable ancestral characters. C. macropterus has 2n = 50, 20m+20sm+10a (fundamental number [FN] = 90), simple interstitial nucleolar organizing regions (NORs), and four 5S rDNA sites terminals, two in synteny with the 18S rDNA. P. eigenmanni has 2n = 56, 28m+20sm+2st+6a (FN = 106), simple NORs, and two 5S rDNA sites terminals. E. aguanai has 2n = 56, 36m+12sm+2st+6a (FN = 106) and 18S and 5S rDNA sites interstitial syntenic in the chromosome 1. All species exhibited a higher amount of heterochromatin, differing from the pattern of the family, and strong marking associated with NORs. The integration between molecular phylogenetic data and karyotype data indicated a high probability that 2n = 56 and simple terminals NORs in the short arm are ancestral characters in Pimelodidae, evidenced in "sorubiminae." In the OCP clade derived traits were observed resulting from chromosomal changes that played a critical role in the karyotype evolution of the group. © Copyright 2019, Mary Ann Liebert, Inc., publishers 2019.
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