We analyzed the molecular divergence of 2 separate populations of Cebus apella paraguayanus, recently considered a junior synonym of Cebus cay, and estimated its time of separation from C. apella. Cytochrome b DNA from 23 C. cay from Brazil and 9 from Paraguay showed 24 haplotypes (20 and 4, respectively), accounting for 29 variable sites (19 transitions and 10 transversions), with 40.0%, 26.7%, and 33.0% replacements at first, second, and third codon positions, respectively. Genetic distance between haplotypes averaged 0.5%, with 1.1% between C. cay populations. Phylogenetic reconstructions and median joining separated C. cay from Brazil and Paraguay. Neighbor joining showed C. cay and C. apella as sister groups, although C. cay and C. apella collapsed in maximum parsimony and maximum likelihood topologies. Analysis of molecular variance showed the highest variance component between C. cay populations, and mismatch distribution indicated that this species suffered a recent demographic expansion. Divergence time estimates suggested that the 2 populations of C. cay split in the Pleistocene, a period of repeated glaciation events leading to drastic changes in the vegetation composition of different biomes.