Morphologic, molecular and karyologic analyses of Callicebus lugens (Humboldt, 1811) of known geographic origin supported the proposition that this is a valid species. Morphologic and morphometric analyses showed evident differences between C. lugens and two other related taxa of the same group (Callicebus purinus and Callicebus torquatus). Cytochrome b DNA analyses (maximum parsimony, neighbour joining and maximum likelihood) were congruent in showing a strong association between C. lugens and Callicebus sp. of the torquatus group in one branch and a sister branch further divided into two clades: one with species of the personatus group and another, with species of the moloch group. Karyotypic analysis showed that C. lugens has the lowest diploid chromosome number of the primate order (2n = 16). Comparisons with other congeneric species clearly supported the proposition that C. lugens is karyotypically similar to others of the torquatus group.