Techniques to rapidly identify the basidiomycete fungal partner of ectomycorrhizal associations would be a major advantage for ecological, fungal population dynamics and life history studies of epigeous and hypogeous forms in plantations, forests, wild lands and other native or natural vegetation. PCR–RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) identification of DNA regions is an available technique; however, primers which have a high probability of amplifying only the basidiomycete DNA are needed. Here we have assessed the specificity, sensitivity and discrimination of six different primer pairs, three targeting nuclear and three mitochondrial regions, for use in identification of Australian basidiomycete fungi from Eucalyptus forests by matching PCR–RFLP patterns to morphologically defined species. Two sets of primers, one newly designed and targeting the nuclear ribosomal DNA internal transcribed spacers (ITS) and the other amplifying a fragment of mitochondrial large subunit ribosomal DNA met the requirements of high specificity and sensitivity, amplifying DNA from a broad range of larger basidiomycetes, with no amplification of plant, bacterial or ascomycete DNA. The specificity of the ITS primer pair was compared with that of ITS1-F/ITS4-B. PCR–RFLP of the two regions discriminated fungi to species level for 91 fungal species from 28 families. Hence these two DNA regions and the specific primers are a potential practical PCR–RFLP tool for identifying basidiomycetes associated with plants from field samples.