β-defensins (BD) are the largest family of vertebrate defensins with potent antimicrobial, chemotactic and immune-regulatory activities. Four BD genes (BD1-4) have been cloned previously in rainbow trout but none have been reported in other salmonids. In this study seven BD genes (BD1a-b, 2–4, 5a-b) are characterised in Atlantic salmon and additional BD genes (BD1b and BD5) in rainbow trout. Bioinformatic analysis revealed up to seven BD genes in the genomes of other salmonids that belong to five subfamilies (BD1-5) due to whole genome duplications. BD1-2 and BD4-5 are also present in basal teleosts but only BD1 and/or BD5 are present in advanced teleosts due to loss of one chromosomal locus. BD3 is salmonid specific. Fish BD have a unique three-coding exon structure. Fish BD are highly divergent between subfamilies but conserved within each subfamily. Atlantic salmon BD genes are differentially expressed in tissues, often with low level expression in systemic immune organs (head kidney and spleen) yet with at least one BD gene highly expressed in mucosal tissues, heart, blood and liver. This suggests an important role of these BD genes in innate immunity in mucosa, liver and blood in Atlantic salmon.