Abstract: Populus spp., one of the widely distributed fast-growing perennial tree species globally, is highly susceptible to diseases such as anthracnose. CRISPR/Cas9 technology offers an effective solution to overcome the limitations of conventional poplar breeding, particularly its lengthy cycle, enabling rapid improvement of varietal traits, especially disease resistance. In this study, CRISPR/Cas9-mediated targeted editing was applied to the WRKY33a/b genes in Populus ‘Beilinxiongzhu 2’, successfully generating three distinct double-gene mutant types: Wka, Wkb, and Wkf. Phenotypic analyses revealed that dual-gene editing of WRKY33a and WRKY33b significantly enhanced resistance to （Colletotrichum gloeosporioides Penz.） in the edited lines, with the Wkf lines exhibited improved disease resistance while maintaining normal growth rates and photosynthetic physiology. This study highlights the strategic advantages of CRISPR-based targeted editing in achieving precise modulation of gene expression levels, thereby avoiding growth inhibition associated with overactivation of defense responses. Furthermore, it provides a molecular design framework for developing fast-growing, disease-resistant poplar cultivars.