Biochemical analysis of cytogenetically defined rodent-owl monkey hybrid cell lines segregating owl monkey chromosomes allowed the assignment of 14 structural gene markers to seven owl monkey karyotype V (K-V, 2n = 46) chromosomes. Comparative study of assigned gene loci to chromosomes of owl monkey karyotype V (K-V, 2n = 46) and karyotype VI (K-VI, 2n = 49/50) provides the first evidence supporting some of the previously proposed mechanics of chromosomal rearrangements responsible for the great karyotypic diversity in owl monkeys. In addition, chromosome homologies deduced from gene loci of Aotus and other primates are compared here to determine the conservation or modification of chromosome morphology in the course of primate evolution. The data demonstrate the usefulness of gene mapping as an alternative to banding patterns for obtaining evidence on chromosome evolution.