The persistent reliance on fossil fuels, contributing significantly to global energy consumption and environmental degradation, necessitates alternative renewable energy sources such as biofuels. Sugarcane, a C4 crop with high biomass and sugar content, serves as a prime feedstock for first- and second-generation bioethanol production. This study evaluated ten diverse sugarcane genotypes at the Punjab Agricultural University Regional Research Station, Kapurthala, during 2022–23 to assess morphological and biochemical traits, including stalk length, sugar content, and fibre to infer bioethanol production potential of genotypes. Statistical analyses revealed genotype-dependent variability, underscoring the need for selecting high-yield, cost-effective carbohydrate sources for sustainable bioethanol production. Genotype CoPb18213 demonstrated superior performance in biomass yield and juice quality, making it a promising candidate for integrated first- and second-generation biofuel production. The findings underscore the importance of integrating high-yielding genotypes with efficient crop management and mechanization practices to enhance biomass utilization, reduce post-harvest losses, and improve overall biofuel production efficiency. This study provides valuable insights for the development of sugarcane varieties optimized for sustainable and economically viable bioenergy production.