High demand for edible bamboo shoots ofBambusa tuldaandMelocanna bacciferain many Asian ethnic groups has led to the need for developing intensive bamboo farming. To achieve this,in vitroregeneration of bamboo plantlets is needed due to the long and irregular bamboo flowering cycle and scarcity of bamboo seeds. An effective protocol for plantlets regeneration inB. tuldaandM. bacciferafrom nodal explants following validation of the species using the sequence of trnL-F intergenic spacer region is described. Effective axillary bud breaking was achieved at 3 mg/L of 6-benzylaminopurine (BAP) in MS medium. Importantly, combining 2 mg/L of kinetin (Kn) with 3 mg/L of BAP produced a synergistic effect for shoot multiplication inB. tuldaandM. baccifera. Under optimized conditions in half-strength MS medium supplemented with 3 mg/L of indole-3-butyric acid (IBA), 10 mg/L of coumarin, and 3% sucrose, profuse production of dark-brown rhizome inB. tuldaand abundant rooting (81.67%,,) forM. bacciferawithin 30 days were achieved. The established protocol and the validation of the reported species at the molecular level will be of help to stakeholders in edible bamboo trade to conserve gene-pool and increase productivity.