Magnolia sirindhorniae Noot. & Chalermglin is an endangered species with high ornamental and commercial value that needs to be urgently protected and judiciously commercialized. In this study, a protocol for efficient regeneration of this species is standardized. The lateral buds of the M. sirindhorniae plant were used as an explant. Half-strength Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 6-benzyladenine (BA), 0.1 mg/L α-naphthaleneacetic acid (NAA), and 2.0 mg/L gibberellic acid (GA3) was found to be the optimal medium for shoot induction. The maximum shoot multiplication rate (310%) was obtained on Douglas-fir cotyledon revised medium (DCR) fortified with 0.2 mg/L BA, 0.01 mg/L NAA, and additives. The half-strength DCR medium supplemented with 0.5 mg/L NAA and 0.5 mg/L indole-3-butyric acid (IBA) supported the maximum rate (85.0%) of in vitro root induction. After a simple acclimatization process, the survival rate of plantlets in a substrate mixture of sterile perlite and peat soil (1:3; v/v) was 90.2%. DNA markers were used for assessment of genetic uniformity, confirming the genetic uniformity and stability of regenerated plants of M. sirindhorniae. Thus, the described protocol can safely be applied for large scale propagation of this imperative plant.