Abstract: To apply the good resources of Populus tomentosa in the north of north latitude 41°, and provide high quality seedlings for the construction of ecological protection forest in northern China, the shoots from branch water culture of‘Mengshu No.1’(P. tomentosa hybrid with high cold resistance) were used as explants, the best disinfectant and sterilization time were screened. The stem segments propagation of aseptic seedlings, leaf blade differentiation, rooting culture and vitrified seedling culture were conducted. The shoots were sterilized in 0.10% HgCl for 5 minutes, the disinfectant effect was the best. The pollution rate of explants was 15.60%, and the germination rate was 77.70%. The best proliferation medium of stem segments was MS+ 0.20 mg/L 6-BA+0.05 mg/L NAA, and the average number of differentiation bud was 4.21. The best induction medium of root was 1/2 MS+0.50 mg/L IBA+0.20 mg/L NAA, and the rooting rate was 93.90%. Vitrification seedlings restored best in the medium MS+0.2 mg/L 6-BA + 0.05 mg/L NAA + 30 g/L sucrose+8 g/L AGAR, and the restoration rate was 82.38%. The establishment of in vitro micropropagation system for‘Mengshu No.1’ lays a foundation of somatic polyploid breeding for P. tomentosa, landscaping and ecological construction in northwest China