Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75 %) was observed with BAP at 3 mg l⁻¹. Shoots were multiplied for 30 d on fresh medium with similar composition. A total of 67 % of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81 cm) with BAP (3 mg l⁻¹) alone. Maximum rooting response (87 %) was observed with naphthaleneacetic acid at 0.5 mg l⁻¹. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.