Atlantic and Mediterranean populations of maritime pine are very different in quantitative and adaptative traits, even to be considered different subspecies. Different authors have suggested the existence of glacial refugia for this species both in Portugal and eastern Spain based on molecular markers and supported by pollen and fossil records. In addition, based on the existence of high levels of haplotypic variation in this region, the Castilian Plateau is suggested as a contact zone between Atlantic and Mediterranean gene pools of the species. This hypothesis is tested using Bayesian clustering methods and exclusion tests based on multilocus genotypes obtained by genotyping 13 populations with three highly polymorphic nuclear microsatellites. The results show that Castilian Plateau populations constitute a different gene pool by itself that can not be fully originated from the Mediterranean or the Atlantic gene pools, or from a mixture of both. Therefore, three different hypotheses have been considered as plausible causes to give rise to Castilian Plateau maritime pine populations: 1) fast colonization of the region from one or more Mediterranean refugial areas, 2) existence of cryptic refugia for the species in the zone, and 3) introduction of exotic material by humans.