Three species of Cathartidae (Sarcoramphus papa, Cathartes aura and Cathartes burrovianus) were cytogenetically characterized by G- and C-banding. 18S-28S rDNA was used as a probe to map major ribosomal clusters. These species showed very similar karyotypes, with 2n = 80, 10 pairs of macrochromosomes, a submetacentric Z and a metacentric W chromosome. However, differences were found in the amount and distribution of heterochromatic blocks: S. papa showed heterochromatin only in the pericentromeric region and in chromosome W, while both species of Cathartes had heterochromatic blocks also in the long arm of two acrocentric pairs. Ribosomal clusters were found in a small pair in all three species. Karyotype analysis in Cathartidae revealed that this family has retained similarities to the putative avian ancestral karyotype, and placed Cathartidae in a more basal position in relation to Accipitridae and Falconidae. However, the cytogenetic data still cannot clarify the phylogenetic relationship between this family and other groups, such as Ciconiidae, considered its sister-group according to nucleic acid hybridization studies.