Genetic relationships were investigated among several populations of Pleurotus tuberregium from Nigeria, Papua New Guinea and New Caledonia. Intrastock mating compatibility studies using progeny from two collections demonstrated a tetrapolar mating system for P. tuberregium. Interstock matings among the geographically distinct populations were compatible. All isolates were found to be intersterile with tester strains of other Pleurotus species, showing that P. tuberregium represents a unique intersterility group in Pleurotus. Nucleotide sequences of the ITS region of the rDNA gene were determined for 30 isolates and used to infer phylogenetic structure of populations. Phylogenetic analysis shows that African and Australasian-Pacific isolates form at least two distinct evolutionary lineages. Higher genetic divergence was observed among ITS sequences from the Australasian-Pacific region than among African isolates, which suggests a possible origin of P. tuberregium in the Australasian-Pacific region.