Genetically valuable regions of a species on the basis of previous broad-range genetic surveys must be considered for further studies. This can be especially true in areas with diverse ecological conditions. Southernmost European populations of Scots pine (Pinus sylvestris L.) from the Iberian Peninsula are considered as Tertiary relicts that have persisted throughout Quaternary glaciations in this area, which is regarded as an independent evolutionary unit. Fragmentation and a higher interpopulation genetic variation than that found in northern regions are characteristics of Scots pine Spanish populations. Six chloroplast microsatellite (cpSSR) loci were examined in 324 individuals from 13 Iberian populations in the Northern Meseta region and different approaches for determining population genetic divergence were performed to obtain information for conservation purposes. Very high levels of haplotypic diversity were found in relation to those detected in other pine species from the Iberian Peninsula, providing a new insight into the evolutionary history of different taxa in southern refugia. Most of the genetic diversity is within populations, with very little but significant variation among populations (RST=0.024). Most of variation is due to a few highly differentiated populations, which should be priority candidates for conservation strategies. Big genetic differences were found between closed populations and there was not a clear geographic pattern in the distribution of genetic variation. © Arbora Publishers.