An assessment of the inter- and intraspecific resolution of 5′ nrDNA ITS TRFLP was conducted by computer-simulated restriction analysis of 316 ectomycorrhizal GenBank sequences. Generally, sequences with a similarity of <90% could be distinguished with two to three independent enzyme digests, although sequences with similarity >95% were likely to remain unresolved. Choice of restriction enzyme strongly influenced resolution, especially when less than three enzymes were used. Although our results showed that 5′ nrDNA ITS TRFLP is a powerful tool for distinguishing between species of ectomycorrhizal fungi, closely related species, including species of Rhizopogon, Dermocybe, Hebeloma, and Lactarius produced indistinguishable 5′ nrDNA ITS TRFLP – profiles. 5′ nrDNA ITS TRFLP may also split a species, and the probability of this occurring reflected intraspecific sequence variation. For groups of closely related species, the use of 3′ nrDNA ITS in conjunction with 5′ nrDNA ITS produced improved resolution comparable with RFLP analyses. Overall, our results show that nrDNA ITS TRFLP is a valuable addition to the array of molecular tools available to ectomycorrhizal ecologists. However, simple assessments of ectomycorrhizal species diversity based on numbers of unique TRF created with a single restriction enzyme must be viewed with caution, as for each enzyme we examined, identical TRF common to groups of unrelated species were observed. In addition, to maximize the effectiveness of TRFLP in surveys of ectomycorrhizal fungi it will be useful to combine information from at least two independent enzyme digests in order to distinguish and track species in the field.