ABSTRACTThis study used polymerase chain reaction amplification and direct sequencing of double-stranded DNA to compare the intergenic region between the 3′end of the 26S gene and the 5S gene of the nuclear, ribosomal DNA of several northern hemisphere species ofArmillaria.The aligned sequences were scored for base substitutions and phylogenetic relationships among species were analyzed by parsimony. We concluded 1) that the intergenic region sequences ofA. ostoyae, A. gemina, andA. borealisare more closely related to one another than to any other species; 2) that the intergenic region sequences ofA. lutea, A. calvescens, A. cepistipes, A. sinapina, species IX and species X constitute a closely related group distinguishable from those ofA. ostoyae, A. boralis, andA. geminaby a significant number of substitutions; and 3) that the morphological divergence inA. melleaand Atabescensrelative to the majority of northern hemisphere species and to each other has been roughly paralleled by divergence in the intergenic region, which aligned poorly in all interspecies comparisons includingA. melleaandA. tabescens.A byproduct of this study was the identification of several substitutions in the intergenic region sequence that could potentially provide the basis for rapid species diagnosis.