Two hydrophobin genes (HAH1 and HAH2) have been identified in a Heterobasidion annosum infection-stage cDNA-library. Comparisons of their nucleotide and amino acid sequences show similarity to the coh1 hydrophobin from Coprinopsis cinerea and the sc3 hydrophobin from Schizophyllum commune. Both HAH1 and HAH2 display the amino acid consensus pattern of class I hydrophobins, including the spacing of eight conserved cysteine residues. Real-time quantitative RT-PCR showed high expression of both genes in aerial hyphae but low expression in submerged hyphae and during in vitro infection of pine seedlings. Segregation analysis of HAH1 and HAH2 in a defined cross of Heterobasidion annosum localised HAH1 to linkage group 3 but did not positioned HAH2 in the genetic linkage map. Sequence characteristics and expression patterns of HAH1 and HAH2 suggest a role in aerial growth of mycelia, but not during pathogenesis.