Trichomycteridae is a family of small catfish which is widely distributed throughout Southern Central America and South America. Specimens of Trichomycterus brasiliensis collected in the upper São Francisco River Basin in Brasil were studied cytogenetically. All individuals presented 2n=54 chromosomes, including 34 metacentric, 18 submetacentric and 2 subtelocentric chromosomes. A secondary constriction was observed in the interstitial region of the short arm of the chromosome pair number 20, coinciding with the NOR and 18S rDNA. The first metacentric pair is considerably larger than the second metacentric pair and the NORs occur in the pericentromeric position of the short arm of a large submetacentric pair, allowing to place this species in one of the cis-Andean groups already identified of Trichomycterus.