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The current research focuses on the estimation of fibre content in sweet and high biomass sorghum lines for Production of sweet and high biomass sorghum lines with optimized cell wall components for increased biofuel bioconversion yield, through partially replicated experimental design for F3 populations based on BLUPs values; this study was carried out for four seasons (from postrainy 2020 to rainy 2022) to develop F3 populations of sweet and high-biomass bmr sorghum lines. The proximate fibre component analysis was done in the matured F3 populations by drying the plant samples and grinding them into a fine powder. Fibre quality components such as cellulose, hemicellulose, acid detergent fibre (ADF), acid detergent lignin (ADL), neutral detergent fibre (NDF), metabolizable energy (ME), nitrogen, in vitro organic matter digestibility (IVOMD), and ash, were assessed using Near-Infrared Reflectance Spectroscopy (NIRS). ICSV18003 had the lowest acid detergent fibre (ADF) percentage at 37.04%, while it also had the highest acid detergent lignin (ADL) percentage at 3.84%. The ash percentage was 3.73% in SSV84 × N609. Moreover, the bmr transferred lines of the F3 population exhibited higher levels of cellulose and hemicellulose, while lignin and ash content were decreased. This indicates that the bmr6 and bmr12 alleles can be confidently utilized in sorghum breeding for bioenergy production, as they meet the requirements for bioethanol production.