Abstract: The 14-3-3 protein plays an important role in the cellular signal transduction and primary metabolism of plants. To explore the evolutional pattern of the 14-3-3 gene family will facilitate the understanding of its function in woody plants. In this study, twelve 14-3-3 genes were identified from the Populus trichocarpa genome based on bioinformatics. A phylogenetic study showed that Populus 14-3-3 proteins contain both ε and non-ε forms. A recent whole genome duplication event contributed to the expansion of Populus 14-3-3 gene family, among which a total of five paralogous gene pairs were formed. The analysis of Ka/Ks showed that the purifying selection is the predominant evolutionary force acting on the five paralogous gene pairs. RT-PCR revealed expression divergence in twelve Populus 14-3-3 genes. In conclusion, the results indicate that there are functional divergences in the Populus 14-3-3 gene family after gene duplication. 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