Abstract: This paper describes a protocol for high frequency regeneration from young leaflets of peanut (Arachis hypogaea L.) mature embryos, which provides a effective regeneration system for gene transformation. Through orthogonal experiments with different levels of five factors which were TDZ concentration, BA concentration and NAA concentration in the MS medium, germinating days of seeds and subculture medium, we have obtained optimum conditions for regeneration of plantlets with high frequency. The conditions were TDZ 1.0 μmol/L, BA 0.4 μmol/L, NAA 5.0 μmol/L in the MS medium, germination for 4 days and subculture medium MS0. Among the five factors, TDZ was believed to play key role in inducing differentiation of peanut young leaflets while the subculture medium and germination days came next. The BA and NAA concentration were the least important factors in inducing differentiation. The multiple buds developed further into plants and rooted on medium containing 1/2 MS major elements, 2% sucrose and 5.0 μmol/L IBA. After being transferred onto soil the plants flowered and bore fruits.