The ectomycorrhizal fungi resource of Pinus massoniana and Chinese Fir forest had been investigated in the places of Hunan and YunNan Province. There were 17 species belong to 6 families and 9 genera. All the collected ectomycorrhizal fungi were separated by tissue isolation method, and 12 strains were obtained, which belong to 7 genera of 4 families. The preliminary selection results showed that there were 6 strains presented the better culture characteristics, which were Seleroderma polyrhizum Pers., Scleroderma cepa Pers., Calvatia craniiformis(Schw.)Fr., Lactarius hatsudake Tanaka, Lactarius deliciosus(Fr)Gray., Russula puellaris Fr.. Then these 6 strains were cultivated between every two among them, the results showed that the group of LS3/RHG1 was the optimal combination, the next were the groups of LGD-2/La-HZ3 and LS3/La-HZ3. The groups of A3/ La8 showed antagonism to each other, so it was not suitable to be combined for inoculation. These research results will provide the important technical support for expanding propagation of ECM fungi and mycorrhizal technology application in Pinus massoniana and Chinese Fir plantation.