Abstract: The inhibitory effects of four antibiotics (Cefotaxime Sodium, Ceftriaxone Sodium, Cefradine, Ceftazidime) on Agrobacterium tumefaciens LBA4404 and EHA105 were analyzed. The tissue culture plantlets of P.×euramericana and P.×gaixianensis were chosen as the materials to study the effects of kanamycin on leaf differentiation and stem rhizogenesis of P.×euramericana and P.×gaixianensis. Besides, the effects of Cefotaxime Sodium on leaf differentiation and stem rhizogenesis of P.×euramericana also was analyzed. The result showed that Cefotaxime Sodium, Ceftriaxone Sodium, Ceftazidime had antibacterial effect on Agrobacterium tumefactions LBA4404, and the working concentration was 50mg/L. Among them, the antibacterial effect of Cefotaxime Sodium was the best for Agrobacterium tumefactions EHA105, and the effect of Cefotaxime Sodium was the worse. The tolerance of different Populus on kanamycin was similar. For P.×euramericana, the 10mg/L concentration of kanamycin was ideal to screen leaf differentiation culture, while the concentration was 20mg/L in rootingstage. The concentration of kanamycin was 20mg/L when leaf differentiation culture of P.×gaixianensis was screened, and the concentration was 25mg/L in rootingstage. For P.×euramericana, the effect of Cefotaxime Sodium on the leaf differentiation and stem rhizogenesis was small.