Current understanding of the postglacial colonization of Nearctic and Palearctic species relies heavily on inferences drawn from the phylogeographic analysis of contemporary generic variants. Modern postglacial populations are supposed to be representative of their Pleistocene ancestors, and their current distribution is assumed to reflect the different colonization success and dispersal patterns of refugial lineages. Yet, testing of phylogeographic models against ancestral genomes from glacial refugia has rarely been possible. Here we compare ND1 mitochondrial DNA variation in late Pleistocene (16 000–40 000 years before present), historical and contemporary Atlantic salmon (Salmo salar) populations from northern Spain and other regions of western Europe. Our study demonstrates the presence of Atlantic salmon in the Iberian glacial refugium during the last 40 000 years and points to the Iberian Peninsula as the likely source of the most common haplotype within the Atlantic lineage in Europe. However, our findings also suggest that there may have been significant changes in the genetic structure of the Iberian refugial stock since the last ice age, and question whether modern populations in refugial areas are representative of ice age populations. A common haplotype that persisted in the Iberian Peninsula during the Pleistocene last glacial maximum is now extremely rare or absent from European rivers, highlighting the need for caution when making phylogeographic inferences about the origin and distribution of modern genetic types.