We analysed 16 populations of Dactylorhiza majalis subsp. majalis located in the northern Poland, simultaneously utilizing both morphological and molecular data. Genetic differentiation was examined using five microsatellite loci, and morphological variation was assessed for 23 characters. At the species level, our results showed a moderate level of genetic diversity (A = 6.00; Ae = 1.86; Ho = 0.387; FIS = 0.139) which varied between studied populations (A = 2.60-4.20; Ae = 1.68-2.39; Ho = 0.270-0.523; FIS = –0.064-0.355). A significant excess of homozygotes was detected in five population, while excess of heterozygotes was observed in four populations, but obtained values were statistically insignificant. Moderate, but clear genetic differentiation was found (FST = 0.101; P < 0.001). Considering the obtained pairwise-FST and number of migrants, we indicated three population groups (I, II, III), where the first one can be divided into two smaller subgroups (Ia, Ib). These three groups differed in the gene flow values (Nm = 0.39-1.12). The highest number of migrants per generation was noticed in the subgroup Ia (8.58). It indicates a central panmictic population with free gene flow surrounded by peripatric local populations (Ib) with more limited gene flow. Geographic isolation, habitat fragmentation and limited seed dispersal affect limited gene flow among three indicated population groups . A weak but significant pattern of isolation by distance was also observed (r = 0.351; P < 0.05).