Heterobasidion annosum (Fr.) Bref., is a pathogenic hymenomycete which causes white-rot of coniferous trees throughout temperate regions of the Northern Hemisphere. The fungus can be divided into three intersterility groups (IS-groups) in Europe and two IS-groups in North America based on in vitro sexual compatibility and, loosely, on host tree preference. European P, S and F IS-groups prefer pine, spruce and fir respectively, and North American P and S groups prefer pine and fir respectively. This work describes the identification of discriminating characters which reflect underlying genetic differences accumulated between the IS-groups. Two genetic loci in the ribosomal DNA repeat and RFLPs in total genomic DNA were examined. Intraspecific divergence was found in the DNA sequence of PCR amplified internal transcribed spacer region (ITS) in ribosomal RNA repeat unit. It was found that various mutation detection techniques such as RFLP, single strand DNA conformation polymorphism (SSCP), heteroduplex DNA polymorphism and amplification refractory mutation system (ARMS) were applicable for the detection of base variations in the ITS region and therefore for the identification of IS-groups. However, since European S and F strains are genetically closely related to each other, these two were not unequivocally distinguishable. Intergenic spacer region (IGS) in the rRNA repeat unit in H. annosum was also amplified by PCR. The five IS-groups were distinguished by RFLP analysis of the IGS region, though there remained some European S and F group isolates which were also identical at this locus. RFLP in total genomic DNA was seen on ethidium bromide stained agarose gel after electrophoresis and found to be able to differentiate the European IS-groups unambiguously. RFLPs in total genomic DNA revealed with minisatellite probes were also found to be useful for IS-group identification.