Fifty-three isolates of Pisolithus were obtained from various locations in central and eastern Australia. These, along with two isolates from south-east Asia and one from USA, were compared using ITS–RFLP and ITS sequencing analyses. Results of the RFLP analysis initially grouped the isolates into eight RFLP types. ITS sequences were obtained for at least one representative isolate from each RFLP type and compared by a neighbour-joining analysis with Pisolithus ITS sequences available in databases. The majority of isolates clustered into four groups within two major clades, each clade comprising isolates of similar basidiospore characteristics. Most Australian isolates corresponded with recent provisional descriptions of P. albus or P. marmoratus. One isolate (LJ30) had low sequence identity (61·6–78%) to the other isolates and probably represents a separate undescribed Australian species. Significant intraspecific variation was observed in ITS–RFLP profiles for the putative P. albus isolates, suggesting that the sole use of RFLP analysis in diversity assessment may overestimate Pisolithus species richness.