Basidiocarps of Pisolithus associated with indigenous (Afzelia quanzensis Welw.) and introduced (Pinus caribaea Mor. and Eucalyptus camaldulensis Dehnh.) hosts in the lowland forests of the Coast Province of Kenya are morphologically distinct. Genetic variability among 52 Pisolithus basidiocarps, collected beneath the various host plants, was examined based on sequence polymorphism within the internal transcribed spacer (ITS) and intergenic spacer (IGS1) of ribosomal DNA genes. Variability in ITS and IGS1 sequences indicated that the three host-associated morphotypes were genetically different. Consensus trees generated by bootstrap analysis of sequence data of Pisolithus isolates from Australia and Kenya are polyphyletic and strongly suggest that the three different morphotypes/genotypes present in Kenya represent separate biological species. In addition, our data indicate that little genetic exchange occurs in silva between these species.