Genetic variability among isolates of the ectomycorrhizal fungus genus Pisolithus collected in Brazil, USA and France was examined, based on nucleotide sequence of ITS from rDNA genes and restriction polymorphism of the mitochondrial DNA (mtDNA). Alignment of the ITS sequences showed 97.0% homology among isolates from Brazil, 97.8% homology among isolates from USA and France and 75.7–78.5% among the isolates from Brazil and USA/France. These sequences were aligned with ITS sequences of Australian, Brazilian and Kenyan Pisolithus isolates described in the literature. A consensus tree generated by sequence data of all Pisolithus isolates showed the presence of a number of species among the isolates. The mtDNA analysis was carried out by total DNA restriction analysis with different enzymes (Dra I, EcoR I, EcoR V, Hha I–Xba I, Hind III and Rsa I) and probing with purified mtDNA labelled with fluorescein d-UTP. The size of the mitochondrial genome of isolates Pt 90A, Pt 185R and PF cleaved with EcoR I was estimated to be, approx. 40.5, 47.2 and 47.6 kb, respectively. Cluster analysis based on the mtDNA restriction fragments grouped the isolates into two distinct groups, which coincide with their host specificity and geographical origins. PCR-RFLP analysis of mitochondrial rDNA (mt rDNA) from 12 Pisolithus isolates did not show any polymorphism. These data corroborate previous results obtained in our laboratory with RAPD-PCR and rDNA PCR-RFLP markers and support recent taxonomic studies where a number of Pisolithus species were described.