The use of medicinal orchids to treat diseases has been prevalent for centuries. In the present study, genetic diversity of five medicinal orchid species including Orchis mascula (L.) L., Anacamptis coriophora (L.) R.M.Bateman, Pridgeon & M.W.Chase, Himantoglossum affine (Boiss.) Schltr., Dactylorhiza umbrosa (Kar. & Kir.) Nevski, and Ophrys schulzei Bornm. & Fleischm. was investigated using inter-retrotransposon amplified polymorphism (IRAP) molecular markers. In total, 473 IRAP bands were produced that all of them were polymorphic and thus the polymorphism percentage was 100%. Himantoglossum affine species from Javanrood region showed the lowest observed alleles (Na = 0.56) and the lowest effective alleles (Ne = 1.09) among populations, while the highest Na (0.93) and the highest Ne (1.18) were observed in Dactylorhiza umbrosa species from population B of Bast village in Divandareh region. The analysis of molecular variance (AMOVA, P ≤ 0.01) showed intrapopulation variation was 46.00% and inter-population variation was 54.00%. The individuals were classified into seven groups by the UPGMA method and based on the Dice similarity coefficient. Dactylorhiza umbrosa species from population B in Bast village from Divandareh region was more diverse, while Himantoglossum affine from Javanrood was less diverse. The obtained results have an important implication for orchid germplasm characterization, conservation, and improvement.
