The Platyrrhini are one of the most karyologically derived groups of primates and the evolution of their karyotypes is far from understood. The identification of the origin and direction of chromosome rearrangements will contribute to a better understanding of New World monkey phylogeny, taxonomy, and evolution. We mapped homology and identified translocations in the chromosomes of the dusky titi monkey (Callicebus moloch, 2n = 50) and the squirrel monkey (Saimiri sciureus, 2n = 44) by fluorescence in situ hybridization (FISH) of human chromosome paints. The hybridization results established chromosomal homologies between these New World primates, humans, other primates, and more distantly related mammalian species and show that both species have highly rearranged karyotypes. The total number of hybridization signals was 37 in C. moloch and 40 in S. sciureus, which is in the range of most comparisons of human chromosomes with phylogenetically more distant species outside of the primate order. Parsimony analyses of outgroup painting patterns allowed us to propose an ancestral karyotype for New World monkeys consisting of 2n = 56 with homologs to the following human chromosomes or chromosome segments: 1b; 1c; 2a; 2b; 3a; 3b; 3/21; 4; 5; 6; 7; 8a; 8/18; 9; 10a; 10/16; 11; 12; 13; 14/15; 15a; 16a; 17; 19; 20; 22; X; Y. Associations 8/18 and 10/16 are derived ancestral associations for all Platyrrhini. A 2/16 association found in S. sciureus and C. moloch was also seen in Ateles geoffroyi and Cebus capucinus; a 5/7 association in S. sciureus was present in A. geoffroyi, C. capucinus, and Alouatta belzebul. Other associations seen in the dusky titi monkey or the squirrel monkey are probably automorphisms. Comparison with chromosome phylogenies based on R-banding [Dutrillaux et al., 1986] showed that there were many errors in assigning homology with human chromosomes. The chromosomal phylogeny of New World monkeys based on banding patterns is in need of revision using modern molecular methods.