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We propose the first molecular systematic hypothesis for the origin and evolution of Cebus capucinus based on an analysis of 710 base pairs (bp) of the cytochrome c oxidase subunit II (COII) mitochondrial gene in 121 C. capucinus specimens sampled in the wild. The animals came from the borders of Guatemala and Belize, Costa Rica, and eight different departments of Colombia (Antioquia, Chocó, Sucre, Bolivar, Córdoba, Magdalena, Cauca, and Valle del Cauca). Three different and significant haplotype lineages were found in Colombia living sympatrically in the same departments. They all presented high levels of gene diversity but the third Colombian gene pool was determined likely to be the most ancestral lineage. The second Colombian mitochondrial (mt) haplogroup is likely the source of origin of the unique Central America mt haplogroup that was detected. Our molecular population genetics data do not agree with the existence of two well-defined subspecies in Central America ( limitaneus and imitator). This Central America mt haplogroup showed significantly less genetic diversity than the Colombian mt haplogroups. All the C. capucinus analyzed showed evidence of historical population expansions. The temporal splits among these four C. capucinus lineages were related to the completion of the Panamanian land bridge as well as to climatic changes during the Quaternary Period. Am. J. Primatol. 74:366-380, 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]