Abstract: The pathogenicity of Colletotrichum gloeosporioides on fruit of 12 Walnut varieties and leaves of 14 Walnut varieties were determined adopted inoculation methods in laboratory in order to confirm the difference of the pathogenicity of Colletotrichum gloeosporioides on different walnut varieties, and provide the base for controlling the walnut anthracnose. The results showed that: (1) inoculating fungus dish was reliable methods because the disease happened early and spread fast, compared with spraying and directly picking mycelium. (2)The pathogenicities of Colletotrichum gloeosporioides on fruits of ‘Zhonglin 1’, ‘Zha 343’, ‘Jinfeng’ were the strongest, and the diameter of spot >10 mm. The pathogenicity on fruits ‘Zhonglin 5’, ‘Liaohe 1’, ‘Xiangling’, ‘Luguang’, ‘Jing 861’, ‘Jinboxiang 1’ and ‘Liaohe 4’ were the second place, and the weakest was ‘Fenghui’ and ‘Lvbo’. (3)Compared with fruit, leaves had an earlier onset, beginning onset after three days of invasion, the leaf spots of ‘Fenghui’, ‘Liaohe 4’ and ‘Lvbo’ 3 species were the largest, diameter of spot up to about 20 mm, the best performance was ‘Beijing 861’, ‘Liaohe 1’ and ‘Liaohe 3’, the diameter of spot were about 10 mm.