Laetiporus sulphureus causes heart-rot in deciduous trees and conifers. European isolates were examined using nuclear DNA markers as well as somatic and sexual compatibility. Pairing tests for somatic compatibility with isolates from 20 different host individuals revealed that each tree is normally colonized by a different genet. The hyphae are multinucleate, basidiospores are binucleate, and conidia contain 2–6 nuclei. Basidiospore germination was erratic, and no clear mating pattern was observed. In one case all single spore isolates from the same basidioma were compatible with each other. Single spore isolates from coniferous and from broadleaf trees were incompatible. The nuclear ribosomal DNA ITS regions from six isolates of different origin revealed nucleotide distances of up to 14·6%. Phylogenetic analysis resulted in a single clade for all isolates. Within this clade, a subclade including only the isolates from gymnosperm hosts was present, indicating that these isolates were derived from ancestral lines from broadleaf hosts. While there is evidence that more than one species or ecotype is represented within the current definition of L. sulphureus, circumscription of additional taxa, and separations within this taxon, remain unclear.