Amplification of the internal transcribed spacer (ITS) of ribosomal RNA genes using PCR and subsequent RFLP analysis offers promise for improved species-level identification of ectomycorrhizal fungi. Intrageneric homogeneity and intraspecific heterogeneity of these characters are, however, frequent. A diverse group of ectomycorrhizal fungi with worldwide distribution was characterized by ITS-RFLP analysis. DNA of 64 identified and five unknown ectomycorrhizal fungi was extracted and their ITS regions were selectively amplified with ITS1-F and ITS4 ribosomal RNA gene primers. Each PCR product was digested with seven restriction enzymes. The resulting restriction fragments were compared by distance matrix analysis. Restriction enzyme analysis of amplified fragments was sufficient for distinguishing all but two closely related species. Intraspecific variation was observed in Cenococcum geophilum and Pisolithus arhizus, providing further evidence that these taxa represent species complexes.