We investigated the chromosome complement of Rhamdia hilarii using C-banding, silver nitrate and CMA 3 staining, and chromosome digestion with 9 restriction endonucleases (RE). The results revealed that this species has 2n= 58 to 2n= 60 chromosomes, with the numerical variation being due to the presence of 1 or 2 metacentric B-chromosomes. The C-banding pattern showed a small amount of heterochromatin in A-chromosomes and heterochromatin in both telomeric regions of B-chromosomes. The Ag-NOR and CMA 3 staining revealed that the rDNA loci were located in the terminal position on the short arm of a pair of submetacentric chromosomes. The heterochromatin of the B-chromosomes was not stained brightly with CMA 3 showing that these regions segments are not GC-rich. Chromosome digestion with different REs permitted the identification of homologous chromosome pairs in some cases. On the basis of the results, we discuss some aspects of chromosome structure and the origin of the B-chromosomes in Rhamdia.