The study was conducted to develop an efficient protocol for plant regeneration of rough lemon (Citrus jambhiri L.) for further improvement by genetic manipulation. Stem, leaf and root explants from in vitro grown seedling of C. jambhiri were cultured on MS medium supplemented with various concentration of α-Naphthaleneacetic acid (NAA), 2,4-Dichlorophenoxy acetic acid (2,4-D) and 6-Benzyaminopurine (BA) for callus and shoot initiation. MS medium fortified with various concentrations of NAA were used for root formation. The ranges of callus initiation from stem explants of C. jambhiri was 13.33% to 80%, whereas leaf explants showed 13.33% to 56.66% callus initiation; and the root explants showed 6.66% to 36.66% callus initiation. The frequency of shoot regeneration ranged from 13.33 to 70%. The highest frequency of callus initiation and shoot regeneration was observed in MS + 1 mg/L 2,4-D + 0.5 mg/L BA and MS + 0.5 mg/L NAA + 3 mg/L BA, respectively. Regenerated shoots showed rooting frequency ranges from 6.66% to 96.66%. The acclimatized plants transferred to field condition and they were survived at 100% frequency and showed the original phenotypes. MS with 1 mg/L 2,4-D and 0.5 mg/L BA is the proper medium for high frequency (80%) callus induction in C. jambhiri and the stem explant showed the highest callus initiation frequency. MS supplemented with 0.5 mg/L NAA + 3 mg/L BA and MS with 0.2 mg/L NAA are the best media for high frequency shoot regeneration (70%) and root initiation (96.66%), respectively. [Fundam Appl Agric 2021; 6(4.000): 359-367]