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<i>Phlebiopsis gigantea</i> (Fr.) Jülich is a well-known generalist conifer wood saprobe and a biocontrol fungus used in several world countries to prevent stump infection by tree pathogenic <i>Heterobasidion</i> fungal species. Previous studies have reported the presence of regional and continental genetic differentiation in host-specific fungi, but the presence of such differentiation for generalist wood saprobes such as <i>P. gigantea</i> has not been often studied or demonstrated. Additionally, little information exists on the distribution of this fungus in western North America. The main purposes of this study were: (I) to assess the presence of <i>P. gigantea</i> in California, (II) to explore the genetic variability of <i>P. gigantea</i> at the intra and inter-continental levels and (III) to analyze the phylogeographic relationships between American and European populations. Seven loci (nrITS, ML5–ML6, <i>ATP6</i>, <i>RPB1</i>, <i>RPB2</i>, <i>GPD</i> and <i>TEF1-α</i>) from 26 isolates of <i>P. gigantea</i> from coniferous forests in diverse geographic distribution and from different hosts were analyzed in this study together with 45 GenBank sequences. One hundred seventy-four new sequences were generated using either universal or specific primers designed in this study. The mitochondrial ML5–ML6 DNA and <i>ATP6</i> regions were highly conserved and did not show differences between any of the isolates. Conversely, DNA sequences from the ITS, <i>RPB1</i>, <i>RPB2</i>, <i>GPD</i> and <i>TEF1-α</i> loci were variable among samples. Maximum likelihood analysis of <i>GPD</i> and <i>TEF1-α</i> strongly supported the presences of two different subgroups within the species but without congruence or geographic partition, suggesting the presence of retained ancestral polymorphisms. <i>RPB1</i> and <i>RPB2</i> sequences separated European isolates from American ones, while the <i>GPD</i> locus separated western North American samples from eastern North American ones. This study reports the presence of <i>P. gigantea</i> in California for the first time using DNA-based confirmation and identifies two older genetically distinct subspecific groups, as well as three genetically differentiated lineages within the species: one from Europe, one from eastern North America and one from California, with the latter presumably including individuals from the rest of western North America. The genetic differentiation identified here among <i>P. gigantea</i> individuals from coniferous forests from different world regions indicates that European isolates of this fungus should not be used in North America (or vice versa), and, likewise, commercially available eastern North American <i>P. gigantea</i> isolates should not be used in western North America forests. The reported lack of host specificity of <i>P. gigantea</i> was documented by the field survey and further reinforces the need to only use local isolates of this biocontrol fungus, given that genetically distinct exotic genotypes of a broad generalist microbe may easily spread and permanently alter the microbial biodiversity of native forest ecosystems.