Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of the first intergenic region (IGR1) of nuclear ribosomal DNA was used to clarify the relationship between IGR1 variations and six Japanese biological species of Armillaria: A. gallica, A. nabsnona, A. ostoyae, A. cepistipes, A. mellea and Nagasawa's E (Nag. E: taxonomically unknown species). The procedure was then used to identify Armillaria species associated with Wynnea species (W. americana and W. gigantea) and Entoloma abortivum. By combining the RFLP patterns obtained using three endonucleases, HaeIII, HinfI and MspI, the IGR1s from 18 isolates of six Armillaria species were assigned to nine different RFLP phenotypes and the six species were distinguished from each other. Each of the RFLP phenotypes from the Armillaria isolates associated with Wynnea species or E. abortivum matched a corresponding phenotypes observed among the six Armillaria species. Based on this, all four isolates from W. gigantea were identified as A. mellea, two from W. americana as A. cepistipes, and all three from E. abortivum as Nag. E. These results provide new information on the biological species of Armillaria associated with Wynnea and E. abortivum.