Interspecific and intraspecific variation in the internal transcribed spacer (ITS) region of rDNA of ectomycorrhizal fungi of 44 species in 17 genera were examined using the restriction fragment length polymorphism (RFLP) method. For each species, two to five herbarium vouchers (mainly basidiocarps), collected throughout Fennoscandia, were examined. The ITS region was amplified using the polymerase chain reaction (PCR) and the universal primers ITS1 and ITS4, and subjected to RFLP analysis with three endonucleases. Intraspecific polymorphisms in the ITS region were found in seven species (in nine of the 132 herbarium vouchers). Polymorphisms were due to length mutations, ranging from 5 to 15 bp in four of the seven polymorphic species and mutations in endonuclease restriction sites in six species, mostly affecting only one endonuclease, but in two species two endonucleases. Using a single endonuclease, a unique RFLP pattern could be obtained for more than half the investigated species. By combining different endonucleases, 34 (77%) of the species could be distinguished from another. The remaining RFLP types occurred in one genus. On the basis of the low intra- but high interspecific variation in the ITS region, it is concluded that most ectomycorrhizas formed by the 44 investigated species should be recognized by comparison with this dataset, if the mycorrhizas are sampled from a site located in Fennoscandia. However, in datasets from even larger geographical areas encompassing a higher degree of intraspecific variation in the ITS region, or when mycorrhizas from several sites distant from each other are compared, it might be necessary to include local reference species.