Abstract: Present study is set to develop a method to proliferate and rooting the Persian walnut (Juglans regia L.) in in vitro condition. To evaluate the proliferation of three genotypes, the DKW basal medium supplemented with four different concentrations of BAP (0, 0.5, 1 and 1.5 mg L–1) and IBA (0.01 mg L–1) conducted in three replications under factorial randomized block design were used. The root induction evaluated by using DKW medium supplemented by three different concentrations of IBA (0, 3 and 4 mg L–1) for one week. By study of growth factors, we conclude, the BAP (1.5 mg L–1) have shown appreciable results and the Chandler genotype were more responsive to the concentration compare to Hartley and Z60 genotypes. To root induction and callus initiation, IBA (3 mg L–1) were found to be responsive hormone with 7 days in dark incubation. The Chandler genotype compare to the other genotypes studied here, were more responsive to this hormonal level by showing high root percentage.