We report studies of 14-3-3 genes in rainbow trout, adding to current understanding of the molecular evolution of this multigene family and its functional importance in fish. Ten genes were identified that are apparent duplicates of five ancestors. The duplicated 14-3-3 genes diverged rapidly and their cladogram is markedly different from the phylogenetic tree. The mean rate of nonsynonymous divergence of trout 14-3-3 genes is one order of magnitude greater than that of mammalian genes. An evolutionarily recent genome duplication in salmonid fish relaxed functional constraints, and selection favored establishment of novel isoforms. Differences in tissue distribution of 14-3-3 genes were minor; however, results of 31 microarray experiments showed divergence of expression profiles, which was related to structural divergence of the duplicates. We observed remarkable coordination of expression of all isoforms in our study of stress response in the brain. Profiles of the 14-3-3 genes correlated with a large group of chaperones and genes involved in cell communication and signal transduction. We studied embryonic expression of 14-3-3 genes and found abundant transcripts in the rapidly growing and differentiating parts of embryos such as eyes, tail bud and yolk syncytium during somitogenesis and in gills and pectoral fins after completion of somitogenesis. Consistent expression was observed in the neural crest, which is known to have high morphogenetic potential.