Host specificity of the mycoparasite Hypomyces microspermus to the Xerocomus chrysenteron group has been observed, but primarily from European collections. Our objectives were to test host specificity among Hypomyces spp. associated with boletes in California oak-woodlands, investigate population biology of these parasites, and to initiate studies on host-parasite coevolution. Bolete samples were collected from four locations separated by up to 600 km. Hypomyces isolates were cultured and host tissue samples taken for molecular identification. Based on AFLP analysis, four distinct Hypomyces clades were found with little genotypic diversity within each group. ITS-rDNA regions of selected isolates from each group were sequenced and analyzed along with sequences from a previously published phylogeny. Isolates from two AFLP groups clustered with H. microspermus whereas isolates from the other two AFLP groups clustered with H. chrysospermus. ITS-RFLP followed by sequence analysis identified three bolete hosts: (1) X. dryophilus; (2) a Xerocomus species closely related to X. dryophilus with affinities to X. chrysenteron; and (3) a Xerocomus species related to the X. subtomentosus group, which is not closely related to X. dryophilus and X. chrysenteron. H. microspermus infected X. dryophilus and the species with affinities to X. chrysenteron, whereas H. chrysospermus infected the species with affinities to X. chrysenteron and the species related to the X. subtomentosus group. These results support previous observations that H. microspermus is host-specific to the X. chrysenteron group, and that H. chrysospermus is more of a generalist pathogen. We also conclude that host-parasite coevolution studies within this system will not be possible until a phylogeny of North American boletes is in place.