Centromochlinae is a widely diverse subfamily with more than 50 species and several taxonomic conflicts due to morphological similarity between Tatia and Centromochlus species. However, cytogenetic studies on this group have been limited to only four species so far. Therefore, here we present the karyotype of Centromochlus schultzi from the Xingu River in Brazil using classic cytogenetic techniques, physical mapping of the 5S and 18S rDNAs, and telomeric sequences (TTAGGG)n. The species had 58 chromosomes, simple NORs and 18S rDNA sites. Heterochromatic regions were detected on the terminal position of most chromosomes, including pericentromeric and centromeric blocks that correspond to interstitial telomeric sites. The 5S rDNA had multiple sites, including a synteny with the 18S rDNA in the pair 24st, which is an ancestral feature for Doradidae, sister group of Auchenipteridae, but appears to be a homoplastic trait in this species. So far, C. schultzi is only the second species within Centromochlus to be karyotyped, but it has already presented characteristics with great potential to assist in future discussions on taxonomic issues in the subfamily Centromochlinae, including the first synteny between rDNAs in Auchenipteridae and also the presence of heterochromatic ITSs that could represent remnants of ancient chromosomal fusions.