Aconitum chasmanthum Stapf ex Holmes, a highly valued medicinal plant, is a critically endangered plant species with restricted global distribution. Because there is no published report on the in vitro micropropagation of A. chasmanthum, the present study was undertaken to contribute to the development of an efficient micropropagation protocol for its conservation. Seeds collected from the wild showed enhanced germination after being given a chilling treatment (−4 °C and −20 °C) for different durations (10, 20, 30 and 40 days). Seeds given a chilling treatment of −4 °C for 10 days showed enhanced germination rates of 47.59 ± 0.53% with a mean germination time of 10.78 ± 0.21 days compared to seeds kept at room temperature when grown in an MS basal medium. Nodes, leaves and stems, taken from 20–40-day-old seedlings, were used as an explant for micropropagation. An MS medium supplemented with different concentrations of cytokinins (BAP, Kn), auxins (2,4-D, NAA), and an additive adenine sulphate were tested for callusing, direct shoot regeneration and rooting. Only nodal explants responded and showed direct multiple shoot regeneration with 7 ± 0.36 shoots with an elongation of 5.51 ± 0.26 cm in the MS medium supplemented with BAP 0.5 mg/L, and with a response time (RT) of 10.41 ± 0.51 days and a percentage culture response of 77.77 ± 2.77%. Rhizome formation was observed after 8 weeks, with the highest culture response of 36.66 ± 3.33% in the MS basal media with an RT of 43.75 ± 0.50 days. These rhizomes showed a 60% germination rate within 2 weeks and developed into plantlets. The present in vitro regeneration protocol could be used for the large-scale propagation and conservation of A. chasmanthum. © 2021 by the authors.