Fifteen gene loci for constitutive enzymes previously localized to specific owl monkey chromosomes of karyotypes III, V, and VI are confirmed by their assignments to homologous chromosomes of owl monkey karyotypes I, II, IV, and VII. The syntenic mapping of LDHA and GPI on a large metacentric, II–2, and the separate assignment of these two loci to two acrocentrics, I–9 and I–15, provide genetic evidence supporting the proposed fusion-fission event that characterized the karyotypic difference between owl monkeys inhabiting Colombia and the Panama Canal Zone. Moreover, the proposed hypothesis on chromosome polymorphism among the Colombian owl monkeys with karyotypes II, III, and IV, resulting from a fusion-fission event involving one metacentric and two subtelocentric pairs, is supported by the assignment of LDHB and MDH1 to the large metacentric I–2 and the separate localization of these two gene loci to II–13 and II–14, respectively.