Swertia chirata is an endangered Gentian species used as herbal medicine for various health ailments including liver disorders, malaria, and diabetes. The depletion of S. chirata from the wild for such applications is a concern. Slow rates of propagation because of poor seed germination and low seed viability are presently limiting factors for its large-scale commercial cultivation. For commercial plantation and conservation of existing germplasm, in vitro multiplication is an attractive solution. The present investigation has achieved production of genetically uniform plants from the nodal explants. Shoot regeneration was obtained in shoot-inducing medium containing half-strength Murashige and Skoog's basal medium supplemented with 0.44 μM 6-benzylaminopurine and 4.65 μM 6-furfurylaminopurine. The highest number of shoots, at 18 per explant, regenerated when media was further fortified with 10 mM KNO₃ and 75 mg l⁻¹ of casein hydrolysate. Tissue culture regenerated plantlets were successfully transferred to the field and produced viable seeds. Studies of chromosome number and a comparative analysis of the DNA fingerprinting profiles indicate genetic stability of the regenerated plants.