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Serine/arginine-rich (SR) proteins are important splicing factors in plant development and abiotic/hormone-related stresses. However, evidence that SR proteins contribute to the process in woody plants has been lacking. Using phylogenetics, gene synteny, transgenic experiments, and RNA-seq analysis, we identified 24 <i>PtSR</i> genes and explored their evolution, expression, and function in <i>Popolus trichocarpa</i>. The <i>PtSR</i> genes were divided into six subfamilies, generated by at least two events of genome triplication and duplication. Notably, they were constitutively expressed in roots, stems, and leaves, demonstrating their fundamental role in <i>P. trichocarpa</i>. Additionally, most <i>PtSR</i> genes (~83%) responded to at least one stress (cold, drought, salt, SA, MeJA, or ABA), and, especially, cold stress induced a dramatic perturbation in the expression and/or alternative splicing (AS) of 18 <i>PtSR</i> genes (~75%). Evidentially, the overexpression of <i>PtSCL30</i> in <i>Arabidopsis</i> decreased freezing tolerance, which probably resulted from AS changes of the genes (e.g., <i>ICE2</i> and <i>COR15A</i>) critical for cold tolerance. Moreover, the transgenic plants were salt-hypersensitive at the germination stage. These indicate that <i>PtSCL30</i> may act as a negative regulator under cold and salt stress. Altogether, this study sheds light on the evolution, expression, and AS of <i>PtSR</i> genes, and the functional mechanisms of <i>PtSCL30</i> in woody plants.