The sexual system of Heterobasidion insulare was elucidated and 40 specimens originating from the mainland of China, Taiwan, and Japan were studied with the aid of mating tests. Most of the material was also investigated with DNA fingerprinting using random amplified microsatellite (RAMS) and M13 minisatellite primers. Denaturing gradient gel electrophoresis (DGGE) and sequencing were applied for detecting variation in the internally transcribed spacer (ITS) region of the ribosomal DNA cluster. H. insulare proved to be a species complex with bipolar mating system. Clamp connections occurred in both heterokaryotic and homokaryotic mycelia. Three intersterility groups were found: the ‘T group’ included specimens from Taiwan and Guizhou Province (southern China), the ‘N group’ specimens from northern China and Japan, and the ‘Y group’ specimens from Yunnan (south-west China). The T group seems to be totally intersterile with the two other groups, and was grouped separately in neighbour joining analysis based on DNA fingerprints. As concluded from mating experiments, groups N and Y are partially intersterile, but they are able to produce hybrid heterokaryons, no group specific DNA markers were found, and they were grouped together in neighbour joining analysis. The identity of H. insulare sensu stricto was studied from the type collections. It seems that none of the above-presented taxa can be referred to it, and we propose the term H. insulare sensu lato to be used in future studies.