The study presents a phylogenetic analysis of species of the moss genus Orthotrichum. ITS1 and ITS2 for 30 species were sequenced. The results do not fully reflect the current division of Orthotrichum into subgenera and sections. Molecular data divide the genus into two groups of species with superficial and immersed stomata, and indicate a clear distinctness of dioecious species. This suggests that a previous concept postulating that the subgenus Orthophyllum should be excluded from the genus Orthotrichum might be justified.