Paeonia emodi Wall. ex Royle, a valuable medicinal and ornamental species native to the north-western Himalayas, is threatened by overharvesting and habitat loss [1, 2]. This study presents a steadfast in vitro regeneration protocol for its conservation and mass propagation. Nodal explants were cultured on Murashige and Skoog’s (MS) basal medium with various concentrations of plant growth regulators (PGRs). Among PGRs, 2.0 mg L⁻¹ BAP and 2.5 mg L⁻¹ zeatin, individually, induced the maximum shoot induction rates (70.44% and 59.66%) with 8.70 and 5.17 shoots per explant, respectively. A combination of 2.5 mg L ⁻¹ zeatin and 1.5 mg L⁻¹ TDZ resulted in 66.20% shoot induction, improved shoot length (6.63 cm), and faster response (28.13 days). The highest rooting efficiency (48.22%) was obtained on half-strength MS medium containing 0.25 mg L⁻¹ NAA and 0.5 mg L⁻¹ IBA. Acclimatized plantlets showed 80–85% survival under greenhouse conditions. This simple and reproducible protocol supports large-scale propagation of P. emodi, and lays the groundwork for its conservation, genetic improvement, and commercial use.