Banana (Musa sp) peels, usually considered agricultural wastes have been identified as a valuable bioresource for non-food industrial applications. This study combined DNA barcoding and biochemical analysis was combined to assess peel oil from four Musa cultivars in Enugu State, Nigeria. Taxonomical identification using RbcL gene classified the cultivars as Musa ABB group/Ukpoko nsukpo (cooking), Musa acuminata AAA/Une (dessert), Musa AAB/Une nsukpo (silk banana) and Musa AAB/Ukpoko (horned plantain). Oils extracted using Soxhlet method were analyzed for physicochemical properties including fatty acid composition using GC-MS. Oil yields ranged from 7.4% to 14.4%, with the highest yield in Musa ABB group/Ukpoko nsukpo. High kinematic viscosity and saponification values shows oil is suitable for soap and biolubricant production, while fatty acid profiles rich in oleic, palmitic, linoleic, and lauric acids support applications in biofuels, cosmetics, and other bio-based products. ANOVA demonstrated cultivar-specific differences in fatty acid content (p < 0.05) and phylogenetic analysis confirmed genetic divergence among cultivars. This is the first research in the region applying this integrated approach to provide insights linking genetic identity to biochemical diversity. Results from this study demonstrates the potential of banana peel as a sustainable raw material for industrial valorization, support local economy and agro-waste valorization