There is a controversy regarding how many species the genus Lagothrix contains, since the Lagothrix lagothricha subspecies have been recently proposed to be actual species. Clarification of species status is of particular importance in the case of L. l. lugens, because it is the most endangered and its distribution is restricted to the Colombian Andes, a highly deforested region. Using cytogenetic and molecular markers, we obtained evidence indicating that the subspecies status is appropriate for the two taxa occurring in this country. We also report high levels of intraspecific variability in the karyotype. We find evidence for a late Pleistocene separation of the subspecies, and we propose it is the limited area of contact between the taxa that allowed for them to partially differentiate. Am. J. Primatol. 72:1131–1140, 2010. © 2010 Wiley-Liss, Inc.