The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71–72%) but low identities to mammalian T-bet genes (41–42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73–88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis.