The patterns of genetic diversity in 14 Cypripedium calceolus populations were investigated in two distant regions, located inside its continuous range (Biebrza valley, NE Poland) and close to the periphery of its range (Alps, SE France). The genetic variation at the species level was found to be relatively high (P% = 37.5%, H_o = 0.145) as compared with that observed in other endangered or rare species. The mean genetic diversity for both European regions did not differ significantly, except for P% (42.5% for Biebrza valley, 34.1% for Alps, p < 0.001). The Biebrza valley yielded almost twice as many genotypes, but genotypic diversity (as measured by G/N and Ĝ/N) was higher in populations from the Alps. This could mean that asexual reproduction is more intense in populations in the Biebrza valley than in the Alps. The results from PCA, UPGMA and AMOVA analyses showed clear genetic differences in C. calceolus between the two European regions. The Mantel test showed positive and significant associations between genetic and geographic distances among populations situated in the Biebrza valley (r² = 0.37, p < 0.05), but not among the Alpine populations (r² = -0.25, p > 0.05). Our data showed that substantial loss of genetic diversity in C. calceolus most likely occurs rather at the population than at the species level.