Shoot tips from two different cultivars of banana known as Grand Naine (AAA) and Elakki (AB), were evaluated for their in vitro response in different concentrations of cytokinin and auxin which included 6-benzylaminopurine (6-BAP; 2, 3, and 4 mg L−1) and α-naphthalene acetic acid (NAA; 1 mg L−1) which was added to Murashige and Skoog (MS) basal media. The mean survival rates for cultured Grand Naine and Elakki explants in the varied growth regulator treatments were 66.25% and 52.5%, respectively. Rooting was initiated in the seventh passage using half-strength MS medium supplemented with 200 mg L−1 activated charcoal and 20 mg L−1 adenine sulfate (AS). The highest mean number of shoots generated per explant at the end of the seventh subculture from Grand Naine was 20.10 ± 0.73, and in Elakki, it was 11.20 ± 0.53. MS media supplemented with 2 mg L−1 6-BAP, and 1 mg L−1 NAA was found to be the best combination for shoot proliferation in both the cultivars. The in vitro-regenerated plants were hardened primarily under shade net conditions using cocopeat substrate and were later transferred to a 1:1:1 ratio of cocopeat, red soil, and sand mix before exposure to field conditions. The survival rate for Grand Naine and Elakki cultivars was 98.5% and 96.5%, respectively, during the acclimatization period of the micropropagules under shade net conditions. Polymorphism was not observed between the mother plant and the in vitro-regenerated micropropagules when analyzed for specific randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR).