Abstract: In order to lay a certain foundation for the improvement of pear tree cultivars, the genetic transformation and the functional verification, Pyrus betulaefolia cotyledon explants were taken as test materials. This research studied the effects of basic culture medium, plant growth regulator, dark incubation time, carbon source, explants and other factors on the regeneration ability, screened out suitable conditions for the cultures of the adventitious bud differentiation and the rooting of Pyrus betulaefolia, and established the regeneration system for Pyrus betulaefolia cotyledon. The results showed that: the best culture medium in which cotyledon induced the growth of adventitious buds was NN69+6-BA 5 mg/L+IBA 0.05 mg/L+sucrose 35 g/L, and its regeneration frequency was 100%; after budding, the best subculture medium was MS+6-BA 1.0 mg/L+NAA 0.1 mg/L, and its average bud regeneration number was 4.84; the best culture medium inducing adventitious buds to take root was 1/2 MS+IBA 1.0 mg/L+sucrose 30 g/L, and the rooting rate was 80%. By optimizing the above conditions, this research had established the efficient regeneration system for Pyrus betulaefolia cotyledon.