We examined the effect of genotypes, explant types, combinations and concentrations of growth regulators on sesame callus induction and shoot regeneration with the cultivars Sesamum indicum L., 2n=26; the wild species S. radiatum, 2n=64, and S. schinzianum, 2n=64; and their distant hybrid strain S. schinzianum × S. indicum. We established the technical system of sesame callus induction and shoot regeneration with high frequency. Among the growth regulators, BA and NAA are suitable for inducing compact green callus, which gives a high differentiation frequency. The optimal medium for sesame callus induction and differentiation was MS basal medium supplemented with 0.1 mg·L^–1 NAA, 2.0 mg·L^–1 6-BA and 30 g·L^–1 sucrose. With this medium, the ratios of the highest level of callus induction and differentiation were 97.50% and 94.02% respectively, which were obtained from wild sesame hypocotyl explants, with the corresponding frequencies of 40.60% and 8.16% for cultivar hypocotyl explants. For the immature embryo explants from the distant hybrids, we achieved the maximum ratios of callus induction (46.67%) and differentiation (89.29%) with this medium. These results form the basis for both germplasm conservation and transgene studies in sesame.