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<i>Heterobasidion annosum sensu lato</i> is a species complex of pathogenic white-rot wood decay fungi which cause root and butt rot in conifer and hardwood species across the Northern hemisphere. Annual losses to forest managers are valued in the billions of dollars, due to tree mortality, reduction in timber yield, and wood decay. In North America, <i>H. irregulare</i> and <i>H. occidentale</i> have a partially overlapping host and geographic range, cause similar disease symptoms and produce similar fruiting bodies, making discrimination between the two of them often difficult. We developed two sets of primers that bind specifically to conserved, but species-specific portions of glyceraldehyde 3-phosphate dehydrogenase and elongation factor 1&#945; alleles. The method is sensitive enough to detect either species from infected wood. Analysis of North American isolates has further clarified the distribution of both species on this continent, including the detection of <i>H. irregulare</i> for the first time on ponderosa pine (<i>Pinus ponderosa</i>) and eastern white pine (<i>Pinus strobus</i>) in British Columbia. This method has the potential to be a valuable tool for the detection of the pathogen in exported/imported wood products, as well as for the further identification and assessment of the distribution of North American <i>Heterobasidion</i> species.