Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non-native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real-time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species-specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real-time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real-time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring.Read less <