Aim The Chilean long-tailed snake (Philodryas chamissonis) has a wide distribution across different latitudes and ecosystems in Chile ranging from the south of the Atacama Desert (26° S) to the extremely humid Valdivian temperate rain forest (40° S). Throughout this vast distribution, which is isolated by the Andes mountain range on the east and the Pacific Ocean on the west, there are biogeographical boundaries and large geographical barriers that must have played an important role in the distribution of genetic diversity within this species. This study aimed at elucidating the evolutionary history of P. chamissonis in Chile by analysis of mitochondrial DNA sequences.Location Chile (29°41'-38°23' S).Methods We extracted DNA from 66 tissue samples collected across different latitudes and amplified and sequenced the mitochondrial DNA control region and the NADH dehydrogenase subunit 4 gene for phylogenetic and population analysis.Results Four distinct haplogroups were identified for P. chamissonis. These are highly consistent with a latitudinal geographic pattern, different ecosystems and the increase in topography towards central Chile. Three of the four haplogroups are concentrated in central Chile (33° S latitude) where the highest herpetofaunal diversity of the country is found. The Maipo River acts as historical geographical barrier for the species influenced by Pleistocene glaciation cycles, leading to a marked phylogeographical boundary. A strong population structure was found for the species (Φst = 0.78, P < 0.0001), with a high haplotype diversity (h = 0.97 ± 0.01) and nucleotide diversity (Π = 0.0151 ± 0.0077).Main conclusions At least three evolutionarily significant units (ESUs) were designated for the species, and these should be taken into account for conservation plans. Three of the four haplogroups found within P. chamissonis are already threatened because their distribution along central Chile overlaps with most of the largest cities in the country.