In this study, 123 sweet sorghum (Sorghum bicolor L.) accessions and 50 mutants were examined with diverse stalk soluble sugars, bagasse enzymatic saccharification and wall polymers, indicating the potential near infrared spectroscopy (NIRS) assay for those three important parameters. Using the calibration and validation sets and modified squares method, nine calibration optimal equations were generated with high determination coefficient on the calibration (R-2) (0.81-0.99), cross-validation (R(2)cv) (0.77-0.98), and the ratio performance deviation (RPD) (2.07-7.45), which were at first time applied by single spectra for simultaneous assay of stalk soluble sugars, bagasse hydrolyzed sugars, and three major wall polymers in bioenergy sweet sorghum. (C) 2014 Elsevier Ltd. All rights reserved.