Hypostomusis a diverse group with unclear aspects regarding its biology, including the mechanisms that led to chromosome diversification within the group. Fluorescencein situhybridization (FISH) with 5S and 18S rDNA probes was performed on ten Hypostomini species.Hypostomus faveolus,H. cochliodon,H. albopunctatus,H.aff.paulinus,andH. topavaehad only one chromosome pair with 18S rDNA sites, whileH. ancistroides,H. commersoni,H. hermanni,H. regani,andH. strigaticepshad multiple 18S rDNA sites. Regarding the 5S rDNA genes,H. ancistroides,H. regani,H. albopunctatus,H.aff.paulinus,andH. topavaehad 5S rDNA sites on only one chromosome pair andH. faveolus,H. cochliodon,H. commersoni,H. hermanni,andH. strigaticepshad multiple 5S rDNA sites. Most species had 18S rDNA sites in the telomeric region of the chromosomes. All species butH. cochliodonhad 5S rDNA in the centromeric/pericentromeric region of one metacentric pair. Obtained results are discussed based on existent phylogenies for the genus, with comments on possible dispersion mechanisms to justify the variability of the rDNA sites inHypostomus.