We performed multi-directional chromosome painting in a comparative cytogenetic study of the three Atelinae species<i> Brachyteles arachnoides, Ateles paniscus paniscus</i> and <i>Ateles belzebuth marginatus,</i> in order to reconstruct phylogenetic relationships within this Platyrrhini subfamily. Comparative chromosome maps between these species were established by multi-color fluorescence in situ hybridization (FISH) employing human, <i>Saguinus oedipus</i> and <i>Lagothrix lagothricha</i> chromosome-specific probes. The three species included in this study and four previously analyzed species from all four Atelinae genera were subjected to a phylogenetic analysis on the basis of a data matrix comprised of 82 discrete chromosome characters. The results confirmed that Atelinae represent a monophyletic clade with a putative ancestral karyotype of 2n = 62 chromosomes. Phylogenetic analysis revealed an evolutionary branching sequence <i>{Alouatta {Brachyteles {Lagothrix</i> and <i>Ateles}}}</i> in Atelinae and <i>{Ateles belzebuth marginatus {Ateles paniscus paniscus {Ateles belzebuth hybridus</i> and <i>Ateles geoffroyi}}}</i> in genus <i>Ateles</i>. The chromosomal data support a re-evaluation of the taxonomic status of <i>Ateles b. hybridus.</i>