Abstract: Chemical mutagenesis can effectively create many new peanut genotypes. And appropriate conditions of induced mutation can increase mutation efficiency and develop peanut germplasm resources well. Seed explants of two peanut varieties were used and five concentration (0.3%, 0.6%, 0.9%, 1.2%, 1.5%) and five processing time (0.5 h, 1h , 3h, 5h, 7h) were set up. The results showed that the appropriate combinations of zhonghua No.5 were 0.9% 7h and 1.2% 5h and the appropriate combinations of yuanza 9102 were 0.6% 7h and 0.9%7h according to half-death rates for the selection criteria. The obvious physical injury was caused by EMS treatment and the sensitivity of EMS to two varieties were a little difference. Molecular identification results showed that the DNA mutation had already occurred.