Cladonia rei and Cladonia subulata are morphologically similar, but chemically different cup lichens of dry grasslands and nutrient-poor ruderal habitats. Recently, C. rei has been synonymized with C. subulata on the basis of combined morphological and chemical investigations. However, doubts remained due to a molecular divergent North American sample of C. rei compared to European C. subulata. To clarify the situation, using molecular methods, we analysed chemically different European samples of C. rei and C. subulata, as well as other morphologically or chemically similar Cladonia species. Molecular data show that European and North American samples of C. rei belong to the same clade, which is closely related to C. fimbriata and followed by a subclade with C. coniocraea and C. ochrochlora. The subclade of C. subulata appears to be distinct from C. rei. In concordance with molecular data, the presence of homosekikaic acid is the determining chemical feature for C. rei. In addition, C. humilis and C. innominata proved to be molecularly distinct species.