To systematically study the change of growth, physiological indexes and radiotolerance of Osmanthus fragrans Branches, three species of osmanthus were subjected to 60Co-γ radiation. Four different radiation dose (15、25、35、45 Gy) were conducted to study the influences of 60Co-γ radiation on the morphology of radicle and spear of three O. fragrans (i.e., O. fragrans jingui, O. fragrans yingui, O. fragrans dangui) and DNA, ascertain the medial lethal dose (LD50), the branches without irradiation treatment were taken as control. The results showed that the number and length of roots, soluble sugar and soluble protein content in the buds of three species of O. fragrans were negatively correlated with radiation dose. LD50 dose for branches of O. fragrans jingui, O. fragrans yingui and O. fragrans dangui were 39.77、32.19、 27.96 Gy respectively, indicating the radiotolerance of O. fragrans jingui is the highest, followed by O. fragrans yingui, and O. fragrans dangui is the lowest. This research can provide evidence for the selection of suitable radiation dose for Osmanthus fragrans mutation breeding and also provide for a basis for irradiation breeding in O. fragrans.