A phylogeographic study of Callicebus lugens was carried out based on cytochrome b DNA sequence data. Here, we report, for the first time, the distribution of C. lugens south of the Rio Negro, in Barcelos municipality (Amazonas State, Brazil), indicating that this river is not the southern boundary of the distribution of this species as previously proposed. Specimens from the north and south banks showed the same diploid number (2n = 16), while phylogenetic reconstructions based on maximum parsimony, distance, and maximum likelihood analyses grouped all specimens in a strongly supported clade comprising 2 separate lineages, in coincidence with their geographic distribution along riverbanks. Median-joining analysis showed a similar separation, with 22 transitions between the 2 groups, whereas time of divergence estimates indicated that the splitting of the C. lugens lineages occurred some 2.2 million years before present. Conservation strategies should take into consideration that this species might be sympatric with Callicebus torquatus at the south bank of Rio Negro.