The Chilotan Archipelago and surrounding areas of north-western Patagonia (41°–43°S, 72°–74°W) offer a unique opportunity to study the interplay between the recent genetic and paleoenvironmental evolution on temperate rainforest environments. Previous studies in this region have postulated that land biota persisted west of the Patagonian ice sheet, in ice-free low-elevation regions of the mainland, and the north-western portion of Isla Grande de Chiloé during Quaternary ice ages. In this study, we analysed the phylogeographical structure (Cytochrome b) of the iguanid lizard Liolaemus pictus to estimate their genetic structure in response to glacial–interglacial cycles and colonization routes. We found that populations from the mainland and Isla Grande de Chiloé do not share haplotypes and, thus, are divergent haplogroups. This divergence might reflect an ancient isolation, much older than the last glaciation. Moreover, the existence of four divergent haplogroups among L. pictus populations in the mainland suggests the persistence of multiple isolated populations during the last glaciation. Our results also indicate that the colonization of small islands occurred from several source sites, located both in the mainland and in Isla Grande de Chiloé, after the Last Glacial Maximum.