A new family of antimicrobial peptide homologues termed Sp-Amp has been discovered in Pinus sylvestris (Scots pine). This is the first report of such proteins to be characterized in a conifer species. Sp-AMP1 was identified in a substructured cDNA library of root tissue infected with the root rot fungus Heterobasidion annosum and encodes a mature peptide of 79 amino acid residues. Three additional members of the Sp-AMP family (Sp-AMPs 2–4) encode cysteine-rich proteins of 105 amino acids, each containing an N-terminal region with a probable cleavage signal sequence. Northern analysis confirmed that Sp-AMP expression is elevated in Scots pine roots upon infection with H. annosum. These peptides share 64% amino acid identity with a mature protein from Macadamia integrifolia (MiAMP1), which allowed us to build a homology model for preliminary analysis. Southern analyses further confirmed that several copies of the gene are present in the Scots pine genome. The potential significance of Sp-AMP in the H. annosum–conifer pathosystem is discussed.