Aims: The goal of this research was the development of a PCR- based assay to identify important decay fungi from wood of hardwood tree species in northern temperate regions. Methods and Results: Eleven taxon- specific primers were designed for PCR amplification of either nuclear or mitochondrial ribosomal DNA regions of Armillaria spp., Ganoderma spp., Hericium spp., Hypoxylon thouarsianum var. thouarsianum, Inonotus / Phellinus- group, Laetiporus spp., Perenniporia fraxinea, Pleurotus spp., Schizophyllum spp., Stereum spp. and Trametes spp. Multiplex PCR reactions were developed and optimized to detect fungal DNA and identify each taxon with a sensitivity of at least 1 pg of target DNA in the template. This assay correctly identified the agents of decay in 82% of tested wood samples. Conclusions: The development and optimization of multiplex PCRs allowed for reliable identification of wood rotting fungi directly from wood. Significance and Impact of the Study: Early detection of wood decay fungi is crucial for assessment of tree stability in urban landscapes. Furthermore, this method may prove useful for prediction of the severity and the evolution of decay in standing trees.