Abstract: The Lentinula edodes strain ’Liaofu-4’ was used as test material, the effects of subculture times on mycelial growth characteristics and micromorphological changes were studied by subculture, in order to provide a reference for strain application and the study of the strain degradation in the process of subculture.The results showed that subculture times had a significant effect on the growth characteristics and micromorphology of mycelia, and the isolate parts of the fruiting body and the composition of culture medium affect the growth rate and state of the subcultured mycelia apparently.With the increase of subculture times, the growth rate of mycelia decreased significantly.After subcultured for 40 times, the growth rate of mycelia isolated from the pileus decreased by 35.62% on sawdust medium, and the mycelia isolated from the junction of the pileus and the stipe decreased by 34.05% on PDA,and the mycelial growth sparse.With the increase of subculture times, the number of mycelia branches and clamp-connection decreased gradually, and the width of mycelia clamp-connection isolated from pileus increased gradually.In conclusion, the subculture of L.edodes could cause the decrease of mycelial growth rate, the weakening of mycelial growth state, and the change of micromorphology including the width of mycelia, the width and quantity of clamp-connection.The study of mycelial characteristics showed isolation part from pileus and the junction of pileus and stalk were better choice.