An in vitro plantlet regeneration system has been developed from cotyledon segments of Capsicum chi-nense Jacq. cv. Naga King Chili, a very pungent chili species of India. Rosette-like structures (RLS) wereinduced on the explants which on transfer to medium containing indole-3-acetic acid (IAA) resulted inmultiple shoots in course of time. Agar-based Murashige and Skoog (MS) medium fortified with 18.16 Mthidiazuron (TDZ) was found to be the most suitable medium for RLS induction and shoot formation. Max-imum number of multiple shoots (9.5 ± 0.39) and roots (8.6 ± 0.50), and root length (2.4 ± 0.02 cm) wereobtained from rosette-like structures in medium containing 5.70 M IAA. Combined effect of putrescine(Put) (5.6 M) and TDZ (4.54 M) mediated direct multiple shoot (5.8 ± 0.44) induction. The in vitrorooted plantlets were transferred to glass house for hardening and acclimatization, wherein 90% survivalwas recorded. The hardened plantlets, thus developed were established in soil and bore normal fruitsafter 4 months of transfer. The capsaicin content in matured fruits of in vitro propagated plants wasfound to be 0.05236 g/g d wt (837,760 Scoville Heat Units, SHU), where as in vivo raised plantlets yielded0.0545 g/g d wt (872,000 SHU) respectively.