<p>Banana (<i>Musa</i> spp.) is the fourth most important crop in the developing world and a staple food for millions. India harbours extensive diversity of edible cultivars, especially in the south and northeast, yet its genetic characterization remains limited. In this study, 34 species-specific microsatellite (SSR) primers were screened to assess genetic diversity among 65 banana accessions representing AA, AB, AAA, AAB and ABB genome groups and wild relatives. Thirty-one primers produced distinct and reproducible fragments, yielding 128 alleles (1–8 per locus; mean 4.1) with high polymorphic information content (0.42–0.95; mean 0.78). Expected heterozygosity (He) values ranged from 0.31 (MACV77) to 0.80 (MACV96), with a mean of 0.55. Observed homozygosity (Ho) varied substantially from 0.03 (MABN20) to 0.88 (MACV169), yielding an average of 0.34. Diversity indices (percentage of polymorphic loci, number of observed and effective alleles, Shannon information index, fixation index, expected heterozygosity and unbiased expected heterozygosity) were consistently highest in wild, AA and AAB group and least in AB group. Genetic diversity estimates revealed an average expected heterozygosity (He) of 0.492. STRUCTURE analysis revealed six genetic subpopulations (K = 6), and analysis of molecular variance (AMOVA) attributed 66% of the variance within populations and 34% between them. Principal coordinate analysis (PCoA) plot and unweighted pair group method with arithmetic mean (UPGMA) derived dendrogram based on SSR data grouped the individuals into distinct genetic clusters based on their genetic composition. SSR markers were highly cross-transferable across wild species (mean 83.87%), with five primers amplifying in all tested taxa. These results provide new insights into the genetic diversity and population structure of Indian <i>Musa</i> germplasm and offer genomic tools for targeted breeding and conservation.</p>