A large number of micropropagated plantlets of banana, Musa acuminata var. Nanjanagudu Rasabale (NR), that were developed from axillary shoot bud explants over 10 years ago were screened for genetic variation, if any, using RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers. Of the 4000 in vitro plantlets, 11 were used for screening that involved shoot cultures with distinct variation in morphological characteristics (morphotypes). Similarly, the mother maintained in the field was also subjected for genetic analysis. Out of the 50 RAPD and 25 ISSR primers screened, 30 RAPD and 5 ISSR primers produced totally 424 clear, distinct and reproducible band classes resulting in a total of 5088 bands where the banding patterns for each primer was highly uniform and comparable to the field-grown mother clone from which the cultures had been established. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and applicable for clonal propagation of banana var. NR over a long period. This is the first report on the use of genetic markers to establish genetic fidelity of long-term micropropagated banana using RAPD and ISSR.