Abstract: Background and Objective: Couroupita guianensis (family: Lecythidaceae) is a medicinally important tree distributed in tropics and widely used in traditional medicine. Propagation of this plant species through seeds is not feasible under natural conditions, as the seeds are consumed along with fruit pulp by peccaries, pigs and monkeys when the fruits split open. However, the seedling emergence and growth is greatly affected by abiotic stress like heat and drought. Micropropagation system is required for clonal propagation, germplasm conservation and genetic improvement to satisfy the pharmaceutical demand of this medicinal plant. Therefore, the current investigation was focused to bring out an effective protocol for in vitro shoot multiplication of C. guianensis using cotyledonary nodes. Materials and Methods: An efficient micropropagation protocol has been established for C. guianensis using cotyledonary nodes from one week old in vitro germinated seedlings. Murashige and Skoog’s (MS) medium and Woody plant (WP) medium amended with various combination of cytokinins [6-benzylaminopurine (BAP), kinetin (KIN), thidiazuron (TDZ)] and auxins [indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA)] were evaluated for their efficiency in inducing multiple shoots. Results: The WP medium was superior to MS medium in the multiple shoot induction (~2 folds) where a combination of 3.0 mg L–1 BAP+ 2.0 mg L–1 KIN induced highest number of shoots (11.04±0.17) per explant. The addition of 0.5 mg L–1 TDZ increased the number of shoots per explant (14.28±0.07) with shoot length of 6.18±0.11 cm. Micropropagated shoots rooted on MS medium supplemented with 3.0 mg L–1 IBA produced more number of roots (2.66±0.11) with root length of 2.96±0.06 cm. An increase in the number of roots (5.33±0.16) and root length (5.73±0.35 cm) was observed with the addition of activated charcoal (1.0 g L–1) to the rooting medium. Rooted shoots transferred to quarter-strength liquid MS basal salts produced secondary roots. After 7 days of secondary roots initiation, healthy shoots were transferred to the soil mixture containing sand, red soil and organic manure (1:1:0.5, v/v/v). Monomorphic DNA fingerprinting pattern obtained using RAPD and ISSR markers confirmed the clonal fidelity of micropropagated plantlets. Conclusion: This study promotes the large-scale clonal propagation of C. guianensis for sustainable utilization in traditional medicine and reintroduction into its natural habitats.