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There is a clear requirement to develop sensitive methods for detecting denned isolates of ectomycorrhizal fungi within the complex microbial communities of natural ecosystems and reforestation sites. We present a method that permits the rapid identification of an ectomycorrhizal isolate using enzymatic amplification (polymerase chain reaction) of DNA extracted either from pure cultures or ectomycorrhizas. A set of oligonucleotide primers capable of amplifying full‐length nuclear 17S and 25S ribosomal RNA genes, together with the ribosomal internal transcribed spacer and intergenic spacer, have been designed and could be used for amplifying target sequences from a wide range of ectomycorrhizal genera. Length polymorphism in the amplified rDNA and restriction endonuclease analysis of nearly 6‐0 kbp of amplified rDNA provided useful criteria for the rapid typing of isolates from different genera and species …