Orthotrichum, sensu stricto, is a genus of about 90 species that are mostly adapted to xerophytic environments, occurring on a variety of substrate types, including tree trunks and rock surfaces. However, three species—O. euryphyllum, O. rivulare, and O. sprucei—inhabit seasonally submerged substrates along streams and other water bodies. The morphological similarity and peculiar ecological preferences of these three species have been recognized taxonomically by several infrageneric treatments, in particular as subgenus Rivularium, assuming that the acquisition of the rheophytic lifestyle as a single event. We examined with sequence data from nuclear and plastid markers with the hypothesis that these three species form a monophyletic group. Adaptation to the rheophytic habitat arose at least twice in Orthotrichum and very likely additional times in the subfamily. The subgenus Rivularium, as previously recognized, is not monophyletic and the three rheophytic species should be placed in the type subgenus.