The rainbow trout (Oncorhynchus mykiss) TGF-β1 sequence was one of the first fish cytokines described. Studies of its expression suggest it is constitutively expressed but displays refractory inducibility. Here we describe a second TGF-β1 (TGF-β1b) gene that is novel in several respects. TGF-β1b possesses typical TGF-β features, including a CXC motif and an integrin binding site, a tetrabasic cut site and a mature peptide of 112 amino acids (aa) containing nine conserved cysteine residues. The mature peptide is 83% identical to the first TGF-β1 sequence described in rainbow trout, that we designate TGF-β1a, and relative to TGF-β1a shows higher homology to Atlantic salmon TGF-β1b, zebrafish TGF-β1a, and sea bass,and seabream TGF-β1. The gene organisation of salmonid TGF-β1b genes, as inferred from Atlantic salmon whole genome shotgun contigs, is a 6 exon/5 intron structure with exons 3 and 4 of salmonid TGF-β1a genes apparently fused together. The two trout TGF-β1 genes have a wide distribution in vivo, with highest expression found in immune tissues for both isoforms indicating that TGF-β1 has a predominant role in immunity of fish. Expression of both genes was also seen during the ontogeny of trout, with TGF-β1a relatively constant in expression level but TGF-β1b increasing over time. Immune responses in head kidney (HK) macrophages induced by pathogen associated molecular patterns (PAMPs), pro-inflammatory cytokines, mitogens and pathway activators highly elevated the expression level of TGF-β1b but not that of TGF-β1a. TGF-β1b expression was also increased by polyinosinic:polycytidylic acid (poly(I:C)) and/or lipopolysaccharide (LPS) stimulation in three different trout cell lines studied. Finally we show that TGF-β1b is potentially involved in defense against infection with viral haemorrhagic septicemia virus (VHSV), which had no effect on TGF-β1a expression. Thus, it is likely the TGF-β1b gene represents a copy which fulfils the major immune orchestrating functions of TGF-β1 as seen in other vertebrates.