Abstract: The study is to improve the rate of EMS mutants of Hemerocallis hybrida. The pre selected highly permeable solid medium(mannitol 36g/L+ sorbitol 36g/L+ sucrose 50g/L) was used in the experiment. The callus of Hemerocallis hybrida was treated with osmotic stress at different time span and then induced by 0.1%EMS semi lethal dose. The effect of osmotic stress on the mutagenic effect of EMS was studied. The growth state, survival rate, differentiation rate and seedling growth rate of callus after osmotic stress and EMS mutation were observed and statistically analyzed. And cell membrane permeability and SOD, POD, CAT enzymes were determined. Results：Osmotic stress pretreatment has some effects on EMS mutagenic effect. Osmotic stress enhanced the damage of EMS to callus. The cell membrane permeability and the activity of SOD and CAT enzymes increased, and the activity of POD decreased. Osmotic stress 30min-60min had no significant effect on the ability of callus differentiation,but increase the mutant rate 6.34%-9.27%. Osmotic stress induced lethal damage to callus in 90min. Conclusion ：Before the 0.1%EMS semi lethal dose induced the callus of Hemerocallis hybridai, a high permeable solid medium (mannitol 36g/L+ sorbitol 36g/L+ sucrose 50g/L) was used for 30min-60min stress pretreatment to improve the mutant rate of 6.34%-9.27%.There is no regularity in the duration of osmotic stress and the rate of increase in mutation rate.