The Spanish populations of Juniperus oxycedrus subsp. macrocarpa are restricted to coastal dune habitats of the Mediterranean and Atlantic coasts. Here, we investigate the genetic structure in this taxon and the possible existence of detailed phylogeographic structure associated with the Strait of Gibraltar using data from amplified fragment length polymorphism (AFLP) and plastid markers for 14 populations in three regions: Andalusia, the Valencian Community and the Balearic Islands. Principal coordinate analysis (PCOA) data revealed a lack of clear genetic structure in J. oxycedrus subsp. macrocarpa based on AFLP. The highest AFLP genetic diversity was found in Andalusia. Plastid markers revealed 14 haplotypes, only two of them were shared among the regions. Andalusian populations had the highest number of exclusive haplotypes. Our results support Andalusia as a reservoir of genetic diversity for J. oxycedrus subsp. macrocarpa, and, as for other gymnosperms, this area could be considered as a genetic refugium. The frequent presence of common and exclusive haplotypes in Andalusia might indicate the colonisation of the Spanish Mediterranean coast from the Atlantic populations. Colonisation of these new areas could be promoted by biological factors, such as the existence of long-lived individuals or potential for seed dispersal. Finally, the Strait of Gibraltar did not constitute a natural barrier to the expansion of this species along the coast, as identical haplotypes are found on the Atlantic and Mediterranean coasts.