AbstractIn contrast to the ‘avian-like’ diploid number (2n = 80), most toucans and aracaris (Piciformes: Ramphastidae) have divergent karyotypes, exhibiting a higher 2n. To identify the chromosomal rearrangements that shaped the karyotype of these species, we applied chicken macrochromosome paints 1–10 and 11 microsatellite sequences to the chromosomes of two representative species, Pteroglossus inscriptus and Ramphastos tucannus tucannus. Paints of chicken chromosomes revealed that at least the first five ancestral chromosomes have undergone fissions, and a fusion between a segment of chicken chromosome 1 and a segment from chromosome 3 occurred in both species. The microsatellite sequences were accumulated mainly in the Z chromosome and in several microchromosomes in both species. These results suggest that the genomes of the Ramphastidae have been shaped by extensive fissions and repetitive DNA accumulation as the main driving forces leading to the higher 2n as found in these species. Furthermore, our results suggest that the putative ancestral karyotype of Ramphastidae already had a high diploid number, probably close to 2n = 112, similar to that observed in P. inscriptus and R. t. tucannus.