Banana is an important fruit crop in the tropics and subtropics; however, limited information on biomarkers and signature volatiles is available for selecting commercial cultivars. Clonal fidelity is a major contributor to banana yield and aroma; however, there are no useful biomarkers available to validate clonal fidelity. In this study, we performed the molecular profiling of 20 banana cultivars consisting of diploid (AA or AB) and triploid (AAA or AAB or ABB) genomic groups. We screened 200 molecular markers, of which 34 markers (11 RAPD, 11 ISSR, and 12 SSR) yielded unequivocally scorable biomarker profiles. About 75, 69, and 24 allelic loci per marker were detected for RAPD, ISSR, and SSR markers, respectively. The statistical analysis of molecular variance (AMOVA) exhibited a high genetic difference of 77% with a significant FST value of 0.23 (p < 0.001). Interestingly, the UBC-858 and SSR CNMPF-13 markers were unique to Grand Nain and Ardhapuri cultivars, respectively, which could be used for clonal fidelity analysis. Furthermore, the analysis of banana fruit volatilome using headspace solid-phase microextraction-gas chromatography-tandem mass spectrometry (HS-SPME-GCMS) revealed a total of fifty-four volatile compounds in nine banana cultivars with 56% of the total volatile compounds belonging to the ester group as the significant contributor of aroma. The study assumes significance with informative biomarkers and signature volatiles which could be helpful in breeding and for the authentic identification of commercial banana cultivars.