Nothofagus pumilio is the dominant and almost ubiquitous tree species in mountainous environments of temperate South America. We used two types of molecular markers (cpDNA and isozymes) to evaluate the effects of the Paleogene paleogeography of Patagonia and more recent climatic oscillations of the Neogene on such cold-tolerant species’ genetic makeup. Phylogeographic analysis on sequences of three cpDNA non-coding regions at 85 populations yielded two latitudinally disjunct monophyletic clades north and south of c. 42°S containing 11 and three haplotypes, respectively. This indicates a long-lasting vicariant event due to the presence of an extended open paleobasin at mid latitudes of Patagonia. Also distribution patterns of cpDNA haplotypes suggest regional spread following stepping-stone models using pre-Cenozoic mountains as corridors. Comparable genetic diversity measured along 41 sampled populations using seven polymorphic isozyme loci provides evidence of local persistence and spread from multiple ice-free locations. In addition, significantly higher heterozygosity and allelic richness at high latitudes, i.e. in areas of larger glacial extent, suggest survival in large and isolated refugia. While, higher cpDNA diversity in lower latitudes reflects the complex orogeny that historically isolated northern populations, lower isozyme diversity and reduced FST values provide evidence of local glacial survival in numerous small locales. Therefore, current genetic structure of N. pumilio is the result of regional processes which took place during the Tertiary that were enhanced by contemporary local effects of drift and isolation in response to Quaternary climatic cycles.