Two closely related commercially cultivated oyster mushroom species, Pleurotus pulmonarius and P. sajor-caju have been differentiated by traditional mating experiments as well as analysis of the variable ITS and IGS sequences of the ribosomal gene cluster. Molecular analysis of the variable ITS and IGS regions has allowed neither reliable differentiation between the morphologically similar species P. pulmonarius and P. sajor-caju nor confirmation of species identity of the P. sajor-caju strains CS-32, H-1, and H-2. Analysis of the sexual (mating) compatibility between haploid tester strains of these two species in monokaryon-monokaryon mating experiments has demonstrated complete reproductive isolation between P. pulmonarius and P. sajor-caju, thereby confirming that these are separate species.