Abstract: Background and Objective: Butea monosperma (B. monosperma) (Lam.) Taub. var. lutea (Witt.), an important and threatened medicinal plant of Fabaceae. It is having potential role in certain biological activities and needs immediate conservation. The present study was focused on establishment of an efficient regeneration protocol and to assess genetic stability among in vitro regenerants. Materials and Methods: In vitro propagation was done on Murashige and Skoog (MS) basal medium supplemented with varying combination of growth regulators such as benzyl amino purine (BAP) and thidiazuron (TDZ) with a concentration ranged from 0.5-3.0 mg L–1 in combination with indole-3-acetic acid (IAA) (0.2 -0.5 mg L–1) for shoot induction. For elongation of shoots, MS medium is supplemented with 0.1-1.0 mg L–1 of gibberellic acid (GA3) and for rooting of elongated shoots indole-3-butyric acid (IBA) at 0.5-2.0 mg L–1 was used. For clonal fidelity analysis, ISSR-PCR method was used by extracting total genomic DNA from leaves of individual regenerated plantlet and its mother plant by cetyl triammonium bromide (CTAB) method. Results: Multiplication has been achieved through direct adventitious shoot formation from axillary buds of nodal explants at 2 mg L–1 BAP along with 0.2 mg L–1 IAA. Excised microshoots were elongated on 1.0 mg L–1 of GA3, which enhanced shoot length to 80% within 2 weeks of culture (3.21±0.78). The fully elongated shoots were rooted with a frequency 89.65% on MS medium supplemented with IBA at 1.0 mg L–1. Rooted microshoots were successfully acclimatized with a survival rate of 78%. Conclusion: The present investigation reports a high frequency regeneration system for its conservation and the reliability of current study was achieved by obtaining the true to type in vitro raised B. monosperma plants which showed monomorphic banding pattern with that of mother plant.