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Several studies have reported glacial refugia and migration pathways for different pine species in the Iberian Peninsula, all of them based on a single-species approach. In this paper, chloroplast microsatellites (cpSSRs) are used to compare population genetic structure and diversity estimates for interspecific pairs of populations located along a cline from southwestern (latitude 36°32′ N, longitude 5°17′ W) to northeastern Spain (latitude 42°14′ N, longitude 2°47′ E) in two widely distributed Mediterranean pines,Pinus halepensisMill. andPinus pinasterAit. Some cpSSRs were shared between species, facilitating comparison of levels of gene diversity at the species level and inferences about within and among species differentiation.P. pinastershowed a much higher number of variants (29) and haplotypes (69) thanP. halepensis(20 and 21, respectively). Moreover, genetic diversity estimates for interspecific pairs of populations along the cline were negatively correlated. Three main causes may explain the differences between species in the present-day distribution of genetic diversity: (1) the distribution of genetic variability before the Quaternary glaciations, with an earlier presence ofP. pinasterin the Iberian Peninsula and a late spread ofP. halepensisfrom eastern and central Europe, (2) the location of the Holocene glacial refugia and the migration pathways from these refugia to the present-day range (from northeast to southwest inP. halepensisand from southwest to northeast and northwest inP. pinaster) and (3) the interactions between species during the postglacial spread. [ABSTRACT FROM AUTHOR]