A genetic linkage map of the basidiomycete Heterobasidion annosum, casual agent of root rot in conifers, was constructed from a compatible mating between isolates from the North American S and P intersterility groups. In a population consisting of 102 progeny isolates, 358 AFLP markers were scored. The linkage analysis generated 19 large linkage groups, containing 6 or more markers, which covered 1468 cM. The physical size to genetic distance was approximately 11.1 kbp/cM. Segregation of three intersterility gene loci were analysed through mating of the progeny isolates with three tester strains carrying known intersterility genotypes. The loci for the two intersterility genes (S and P) were successfully located in the map. Segregation of the mating type locus was analysed by backcrossing the progeny isolates with their parental strains. The mating type locus could not be located in the map.