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According to Auchenipteridae initial morphological data, Auchenipterus and Entomocorus have been considered phylogenetically close, and cytogenetic analyses are limited only to Auchenipterus osteomystax. Here are shown the first results for Auchenipterus nuchalis from the Araguaia River and Entomocorus radiosus from the Paraguay River, generated in order to contribute (1) to the investigation of the Auchenipterus chromosomal diversity and (2) at attempt to better understand the phylogenetic relationship of these Auchenipterinae genera, mainly due to the existence of incongruous characters between Entomocorus and Centromochlinae. The two species presented 2n=58 chromosomes and different karyotypic formulas. The heterochromatin distribution was preferentially shown in terminal regions, with interstitial and/or pericentromeric blocks in submetacentric/subtelocentric pairs in A. nuchalis and E. radiosus. Single and terminal AgNORs was confirmed by 18S rDNA for the analysed species, differing from A. osteomystax (cited as A. nuchalis) from the Upper Paraná River. The variation in the number of 5S rDNA between species and its equilocality in E. radiosus suggests the dispersion of the gene associated with the amplification of heterochromatic regions in the interphase, possibly promoted by the Rabl model. The differences found between the species of Auchenipterus can serve as species-specific characters and auxiliary in studies of these taxa, which historically have been wrongly identified as a single species with wide distribution throughout the Neotropical region, but which is actually represented by different species. Furthermore, there are cytogenetic similarities between E. radiosus and members of Centromochlinae like observed with recent morphological and molecular analyses in the family.