Anolis heterodermus is a poorly known, high elevation anole lizard from northern Andes, currently under threat due to habitat destruction. Although it has been suggested that this taxon corresponds to a species complex, different evolutionary lineages have not been identified. We examined phylogenetic relationships between individuals of A. heterodermus from the Eastern and Central Cordilleras of Colombia and other species of the A. heterodermus series using mitochondrial DNA (partial ND2 gene with adjacent tRNA genes and partial COI gene) and nuclear DNA (partial RAG1 gene) sequences and assessed divergence times between these lineages to elucidate their historical biogeography. We performed genetic analyses for two populations from the middle portion of the Eastern Cordillera of Colombia based on the COI gene. We identified three independently evolving evolutionary lineages within A. heterodermus based on two lines of evidence: (i) all phylogenetic analyses showed A. heterodermus to comprise three strongly supported subgroups, being polyphyletic with respect to other recognized species of the A. heterodermus series and (ii) the uncorrected p-distances of the ND2 gene revealed that the divergence between A. heterodermus lineages exceed the divergence between recognized species of the A. heterodermus series. These lineages should be considered as different evolutionary significant units (ESUs) and candidate species. We found that the diversification of the studied lineages dates to the Middle Miocene to Pleistocene, falling within a period of major orogenic and climatic events in northern South America. The population genetic analyses revealed two management units (MUs) in one of the newly reported lineages from the middle portion of the Eastern Cordillera. Due to the accelerated destruction of their natural habitat, steps should be taken to ensure the conservation of the identified ESUs and MUs.