In vitro micropropagation of banana (Musa spp.) cv.virupakshi (Hillbanana) was studied. Suckers were collected from the germ plasm block of Jain R&D (originally established from the suckers from Palani Hills, Tamil Nadu) during summer. The sucker surface sterilized with 1% NaOCl for 30 min gave 100% survival without any contamination. Apical meristems that were isolated and cultured on MS based media supplemented with benzylaminopurine (BAP) 10.0 mg/l and IAA1.0 mg/l gave higher number of shoots (134.3 shoots/explant) within168 days (24 weeks). Kinetin 2.0 mg/l and NAA0.5 mg/l gave early rooting in just five days with 6.6 roots per plant. Observations were recorded after every four weeks up to six sub-culturing. Acclimatization was done in poly house, followed by shade house under 50% light conditions. The hardened plants when shifted to the field showed luxurious growth. The regenerated micro propagated banana plants were tested for genetic uniformity through 13 inter simple sequence repeat (ISSR) markers recommended by NCS-TCP, DBT. Profiles obtained by all the three ISSR primers namely, 834, 840 and 850, respectively exhibited similar banding patterns, which revealed the existence of genetic uniformity in micro-propagated plants.