Mediterranean forests are fragile ecosystems vulnerable to recent global warming and reduction of precipitation, and a long-term negative effect is expected on vegetation with increasing drought and in areas burnt by fires. We investigated the spatial distribution of genetic variation of Arbutus unedo in the western Iberia Peninsula, using plastid markers with conservation and provenance regions design purposes. This species is currently undergoing an intense domestication process in the region, and, like other species, is increasingly under the threat from climate change, habitat fragmentation and wildfires. We sampled 451 trees from 15 natural populations from different ecological conditions spanning the whole species’ distribution range in the region. We applied Bayesian analysis and identified four clusters (north, centre, south, and a single-population cluster). Hierarchical AMOVA showed higher differentiation among clusters than among populations within clusters. The relatively low within-clusters differentiation can be explained by a common postglacial history of nearby populations. The genetic structure found, supported by the few available palaeobotanical records, cannot exclude the hypothesis of two independent A. unedo refugia in western Iberia Peninsula during the Last Glacial Maximum. Based on the results we recommend a conservation strategy by selecting populations for conservation based on their allelic richness and diversity and careful seed transfer consistent with current species’ genetic structure.