<p>In India, presently, there is a gap between the demand and supply of quality planting material of bamboos. <i>Bambusa balcooa</i> is one of the economically important species in high demand for large scale planting in commercial plantations. To ensure the production of quality planting material, selection of superior genotypes of bamboo species is very important. In the present study, the impact of two genotypes, cytokinins and auxins, during in vitro propagation of <i>B. balcooa</i> was researched. Inoculation of nodal segments of two genotypes, BBAL-4 and BBAL-5, was carried out in a liquid Murashige and Skoog (MS) medium, augmented with cytokinins such as 6-Benzyladenine (BA), Kinetin (Kn), Adenine sulfate (ADS), Zeatin (Zn) and Thidiazuron (TDZ) at concentrations of 3.0 and 5.0&#xa0;mgl⁻¹. Genotypes had a significant effect (at <i>p</i> &lt; 0.05) both during culture initiation and shoot multiplication. The interaction of 5&#xa0;mgl⁻¹ Kn and genotype BBAL-5 had a synergistic effect and resulted in the maximum number of shoots (2.77) during axillary bud proliferation. During in vitro shoot multiplication, the highest average number of shoots (3.29) was recorded on 5 mgl⁻¹ Kn with genotype BBAL-5 after 15&#xa0;days and 11.44 after 30&#xa0;days. For in vitro rhizogenesis, half-strength MS medium was enriched with auxins, viz., Indole-3-butyric acid (IBA), Indole-3-acetic acid (IAA), and α-Naphthaleneacetic acid (NAA), both individually and in combinations (5.0 and 10.0&#xa0;mgl⁻¹). The combination of 5 mgl⁻¹ NAA and 10 mgl⁻¹ IBA yielded the highest rooting percentage (43.98%) with a maximum 10.55 number of roots. Using a substrate mix of sand, soil, and farm yard manure (1:1:1; v/v) resulted in 100% acclimatization of the plantlets. These results highlight the importance of genotypes and plant growth regulators for efficient in vitro propagation of <i>Bambusa balcooa.</i></p>