A phylogenetic analysis of species of Didymodon from the Mediterranean area, Macaronesia, and Southwestern and Central Asia is presented. The ITS1, 5.8S rRNA gene, and ITS2 for 30 species have been sequenced, which represent about 25% of the recognized species in the genus. The molecular data confirm the monophyly of Didymodon as clearly separated from Barbula. The transfer of Didymodon giganteus to Geheebia, of D. sinuosus to Oxystegus, and D. australasiae to Trichostomopsis is not supported by the molecular data. The phylogenetic affinities of the species generally do not correspond with the infrageneric classification proposed for the genus based on morphological characteristics. The only monophyletic section is Asteriscium, but only after the inclusion of D. bistratosus.