European black pine (Pinus nigra Arn.) is a widely distributed Mediterranean conifer. To test the hypothesis that fragmented populations in western Europe survived in situ during the last glacial rather than having been re-colonized in the postglacial period, genetic variation was assessed using a suite of 10 chloroplast DNA microsatellites. Among 311 individuals analysed, 235 haplotypes were detected revealing high levels of chloroplast haplotype diversity in most populations. Bayesian analysis using a model of linked loci, with no prior assumption of population structure, assigned individuals to 10 clusters that corresponded well with the six predefined sampling regions, while an analysis carried out at the population level and assuming unlinked loci, recovered the original six sampling regions. This regional structure was supported by a biogeographical analysis that detected five barriers, with the two most significant separating Alps from Corsica and southern Italy, and southern Spain from the Pyrenees. No signals of demographic expansion were detected, and comparisons of RST with pRST suggested that a stepwise mutational model was important in regional differentiation, but not in population-within-region differentiation. These tests support long-term persistence of the species within the six regions. The temporal depth estimate, assuming a high mutation rate in coalescent modelling, placed the deepest split between the Alps and the other regions at about 150 000 years ago, and the most recent split of Pyrenees from southern France at about 30 000 years ago. Taken together, the data suggest that chloroplast DNA is structured in black pine and disjunct populations in western Europe are likely to have been present during the Last Glacial Maximum.