Two Orthotrichum species of the subgenus Orthophyllum were compared with other representatives of this genus using the internally transcribed spacer regions 1 and 2, the chloroplast trnH-psbA region and ISSR and ISJ DNA markers. The applied DNA markers revealed many bands and mutations specific only to O. gymnostomum and O. obtusifolium. A phylogenetic analysis clearly supported the previous concepts postulating that species of the subgenus Orthophyllum should be recognized as separate genus Nyholmiella.