<p>Banana (<i>Musa</i> spp.) is a globally important staple and cash crop where an efficient anther culture protocol has been developed to enable the production of doubled haploid (DH) lines for use in breeding programs and genomic research. This study compared the effectiveness of different culture media Murashige and Skoog (MS), N6, and Nitsch in inducing embryogenic callus formation from anthers of five different banana genotypes across three genomic groups (BB, AA, AAA). Among the media tested, MS medium proved to be most effective for embryogenic callus induction. Among the five genotypes tested, Bhimkol (BB) produced embryogenic callus on MS medium supplemented with 4.0&#xa0;mg L⁻¹ 2.4-Dichlorophenoxyacetic acid (2,4-D) and 1.0&#xa0;mg L⁻¹ each of Indole-3-acetic acid (IAA), and α-Naphthaleneacetic acid (NAA). The callus were sub-cultured three times at regular intervals onto MS medium fortified with 2.0&#xa0;mg L⁻¹ IAA and 0.5&#xa0;mg L⁻¹ 6-Benzyl amino purine (BAP). The resultant shoots were then transferred to MS medium supplemented with 2.0&#xa0;mg L⁻¹ BAP and 0.5&#xa0;mg L⁻¹ IAA to enhance shoot multiplication and elongation. Rooting was successfully achieved by culturing the single shoots on MS medium containing 1.0&#xa0;mg L⁻¹ each of Indole-3-butyric acid (IBA) and NAA. The rooted plantlets were acclimatized successfully, with 60% survival. However the field establishment per cent was hardly 10%. Flow cytometry analysis confirmed that all plantlets regenerated from androgenic callus were diploids. Genetic uniformity of the regenerated plants and its mother plant was further validated using eight simple sequence repeat (SSR) markers. This study represents a pioneering effort in anther culture of banana and provides a foundational protocol for efficient plant regeneration. The developed method holds significant promise for generating androgenic haploids and dihaploids, advancing banana breeding programs, mutation and genomics research.</p>