Four camelid species exist in South America: two wild, the guanaco (Lama guanicoe) and the vicuña (Vicugna vicugna), and two domestic, the alpaca (Lama pacos) and the llama (Lama glama). However, the origin of the domestic species has been a matter of debate. In the present study, variations in chromosome G banding patterns and in two mitochondrial gene sequences have been used to study the origin and classification of the llama and alpaca.-Similar patterns in chromosome G band structure were observed in all four Lamini species, and these in turn were similar to the bands described for camels, Camelus bactrianus. However, fine and consistent differences were found in the short arms of chromosome 1, separating camels, guanacos and llamas from vicuñas and alpacas. This pattern was consistent even in a hybrid guanaco x alpaca. Equivalent relationship showed the complete cytochrome b gene sequences, and the minimum expansion tree of the partial control region sequence, grouping guanaco with llama and vicuña with alpaca. Phylogenetic analyses showed V. vicugna and L. guanicoe as monophyletic groups. Analysis of both gene sequences revealed two clades within vicuña, concordant with the two described subspecies, but the results for guanaco did not confirm existence of the four previously proposed subspecies. The combined analysis of chromosomal and molecular variation showed close genetic similarity between alpacas and vicuñas, as well as between llamas and guanacos. Although directional hybridization was revealed, our results strongly support the hypothesis that the llama would have derived from L. guanicoe and the alpaca from V. vicugna, supporting reciassification as V. pacos. © 2007 Sociedad de Biología de Chile.