Abstract: Background and Objectives: Presently, determination of optimum protocol for callus induction of any plant is an important issue in tissue culture technology. Therefore, the main objective of this study was to find out an optimum protocol for callus induction from in vitro cultured jojoba by determining the optimum explant and the best growth regulators mixture for callus induction. Materials and Methods: The study used three variant explants namely the leaf disks, seeds and nodal segments for callus formation. Different culture media containing basic Murashige and Skoog (MS) medium components supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid as an auxin (2,4-D) and Kinetin (Kin) as a cytokinin with various concentrations ranging from 0.0, 0.5, 1.0 and 2.0 mg L–1 were used. The total number of treatments were 16. The callus was induced from all explants on MS medium containing the lowest concentration of 2,4-D 0.5 mg L–1 with any concentration of Kin. Results: The results showed that nodal segments were the best for callus formation followed by the leaf disks (leaves) and seeds, respectively. While, the best concentration of proliferation and development of the used explant was 2.00 followed in descending order by 1.00, 0.5 and 0.0 mg L–1, respectively. Conclusion: The study find out that the best concentration of 2,4-dichlorophenoxy acetic acid as an auxin (2,4-D) and Kinetin (Kin) as a cytokinin was 2.00 followed in descending order by 1.00, 0.5 and 0.0 mg L–1, respectively for callus induction.