In this study, the regeneration system was established from leave explants of Populus deltoides cl.‘725’. Meanwhile, the buds and rooting seedlings of Populus deltoides cl.‘725’ were screened using hygromycin B. The results indicated that the best disinfection time of leaves was using 75% of the alcohol for 8 s and 0.1% HgCl₂ for 3 min; The best suitable medium of deltoides cl.‘725’ leaves for callus induction was MS+6-BA 0.2 mg·L^-1+2,4-D 1.5 mg·L^-1+sucrose 25 g·L^-1+agar 6 g·L^-1; appropriate induction differentiation medium was MS+6-BA 1.0 mg·L^-1+NAA 0.3 mg·L^-1+KT 0.3 mg·L^-1+ sucrose 25 g·L^-1+agar 6 g·L^-1; the best multiplication media was MS+6-BA 0.3 mg·L^-1+NAA 0.05 mg·L^-1+sucrose 25 g·L^-1+agar 6 g·L^-1; the best medium for rooting was 1/2 MS+ NAA 0.01 mg·L^-1+ IBA 0.7 mg·L^-1+ sucrose 20 g·L^-1+agar 6 g·L^-1; the critical hygromycin B sensitive concentrations for inducing shoots and roots of the poplar were 2.5 mg·L^-1 and 1.5 mg·L^-1, respectively.