Heterobasidion annosumis one of the most aggressive pathogens ofPinusforests in Europe, causing considerable economic losses. To detectH. annosumfor disease diagnosis and control, we developed a loop-mediated isothermal amplification (LAMP) reaction with a primer set designed from the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) DNA sequences ofH. annosum. In our study, this LAMP assay was found to be capable of efficiently amplifying the target gene within 60 min at 63°C. In specificity tests,H. annosumwas positively detected, and other species were negative. The detection limit of this assay was found to be 100 pg·μL-1, and the assay was also successfully tested for use with basidiospore suspensions and wood samples. This study provides a rapid method for diagnosing root and butt rot caused byH. annosum, which will be of use in port surveillance of logs imported from Europe.