<p><i>Gentiana kurroo</i>, generally known as Indian gentian, is a perennial herbaceous species native to the Western Himalayan region and is critically endangered due to slow natural reproduction and extensive overexploitation for medicinal purposes. In the present study, an efficient in vitro regeneration method was developed for <i>G. kurroo</i> via direct and indirect organogenesis. Direct organogenesis from petiole explants was successfully induced on Murashige and Skoog (MS) medium containing 0.1 mg L^− 1 of 1-napthaleneacetic acid (NAA) and 0.5 mg L^− 1 of thidiazuron (TDZ). In addition, direct shoot regeneration and multiplication were assessed using nodal segments cultured on MS medium containing 6-benzylaminopurine (BAP) or kinetin (Kin) alone, in combination with the same cytokinin, or in combination with NAA. The highest shoot multiplication (7.2 ± 1.20 shoots per explant) was obtained on MS medium containing 0.1 mg L^− 1 NAA and 1.0 mg L^− 1 BAP. Root induction was most effective when shoots were transferred to MS medium supplemented with 1.5 mg L^− 1 indole-3-butyric acid (IBA). The regenerated plantlets were successfully acclimatized, achieving a 75% survival rate. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers were employed to confirm the genetic fidelity of in vitro regenerated plantlets. Callus induction was achieved by culturing leaf explants on MS medium supplemented with 0.5 mg L^− 1 Kin and varying concentrations of 0.5-2.0 mg L^− 1 2,4-dichlorophenoxyacetic acid (2,4-D), with the highest fresh weight (1.15 ± 0.32&#xa0;g) of friable, pale-yellow callus obtained at 0.5 mg L^− 1 Kin combined with 1.5 mg L^− 1 2,4-D. For indirect organogenesis, the callus was transferred to a plant growth regulator-free medium to induce shoot bud initiation, followed by maturation into shoots. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that <i>LATERAL ORGAN BOUNDARY DOMAIN16</i> (<i>LBD16</i>) expression was significantly upregulated during callus induction, followed by downregulation during indirect organogenesis. Conversely, persistent <i>SHOOT MERISTEMLESS</i> (<i>STM</i>) expression was observed in both callus and callus with emerging shoot buds, suggesting its role in the maintenance of meristematic competence. These findings provided valuable insights for the conservation and sustainable propagation of this medicinally important species.</p>