A newly identified cyclophilin-encoding cDNA clone was used to design a codominant inherited EST-PCR marker in Norway spruce. The study of the current minor polymorphism revealed a geographically structured differentiation pattern across 17 test populations, showing a slight clinal variation south-north through Europe. Based on the frequency of alleles, isolation-by-distance analysis and the Ewens-Watterson test, we conclude that a selectively neutral random-drift mutation recently occurred within the Alpine population group, thus being responsible for the genetic variation detected. Analysis of tolerant and susceptible subsets of two adjacent Bavarian spruce populations affected by soil-born NaCl pollution, revealed that the cyclophilin marker locus also confirms biased genotype frequencies. Considering an unlinked PCR marker of a ribosomal protein-encoding EST clone, deviations between pooled tolerant subsets and pooled sensitive subsets were proven to be more significant for two-locus homozygous genotypes than for each locus alone. We suggest that both loci are linked to adaptive genomic regions. Their potential to test the feasibility of marker-assisted selection of both NaCl-tolerant and drought resistant tree populations is discussed.