The chromosomes of Brachidontes rodriguezi were analysed by means of direct Giemsa staining, silver staining, fluorescent in-situ hybridization (FISH) with 18S + 28S rDNA probes, replication banding and chromomycin A3 (CMA) and DAPI fluorescence banding techniques. The diploid chromosome number in this species is 32 and the karyotype is composed of two pairs of metacentric chromosomes, 2 pairs of telo/subtelocentric chromosomes and 12 pairs of subtelocentric chromosomes. 18S + 28S rDNA clusters were located on the short arms of the two pairs of telo/subtelocentric chromosomes. The replication band pattern induced in this species facilitates chromosome pairing and differentiation. The nucleolar organizing regions (NORs) replicate late in the S phase and were associated with bright CMA fluorescence and dull DAPI fluorescence, but not all the four NORs showed bright CMA fluorescence in a given cell; intra- and interindividual variability was found for this character.