Inter-simple sequence repeat PCR (ISSR-PCR) was used to develop markers for simple sequence repeat-rich (SSR) regions for investigation of genetic relatedness of Pisolithus isolates collected from eastern mainland Australia. Primers were designed to amplify ten SSR-rich regions and these were used to screen 14 Pisolithus isolates. Two amplified loci showed size polymorphisms among the isolates (regarded as polymorphic), two were monomorphic for all isolates, while the remainder amplified alleles for only some isolates. UPGMA analysis of the alleles for each isolate at each locus together with ITS-RFLP analysis, separated the isolates into two groups. These two groups appear to correspond to isolates that ITS sequence data have previously separated as P. albus and P. microcarpus.