The relationships based on ITS sequences of 48 Hygrocybe s.l. specimens were studied and compared with previously described taxonomic groups. Our specimens formed two well separated genetic groups. The first one includes the species characterized by vivid yellow and red colours, while species belonging to other clades were pallid or pale brown, and in most cases with pink or olive tones. This separation is supported by the presence of muscaflavin pigments among some species referred to Hygrocybe (Bresinsky & Kronawitter 1986). The subgenera distinguished by morphological features can be relatively well recognized on phylogenetic trees, however, the majority of sections were not supported. Variability in the ITS region of Hygrocybe species is unusually high. In some cases sequences differed by more than 25 %, and the lengths of ITS regions also showed large differences. Taxa that were considered as closely related, e.g. the H. conica aggregate, were found to have identical or highly similar sequences. Our results seem to confirm the taxonomic concept of Bresinsky (2008) who proposed the division of the genus Hygrocybe. Hence H. calyptriformis and all examined members of subg. Gliophorus (H. irrigata, H. laeta, H. nitrata, H. psittacina) and subg. Cuphophyllus could be excluded from the genus Hygrocybe s.str. Based on these results further research using DNA markers at the intergeneric level is suggested to revaluate the taxonomy of former Hygrocybe species.