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Cellulose crystallinity is a crucial factor influencing stem strength and, consequently, wheat lodging. However, the genetic dissection of cellulose crystallinity is less reported due to the difficulty of its measurement. In this study, VIS/NIR spectra and cellulose crystallinity were measured for a wheat accession panel with diverse genetic backgrounds. We developed a reliable VIS/NIR model for cellulose crystallinity with a high determination coefficient (R2) (0.95) and residual prediction deviation (RPD) (4.04), enabling the rapid screening of wheat samples. A GWAS of the cellulose crystallinity in 326 wheat accessions revealed 14 significant SNPs and 13 QTLs. Two candidate genes, TraesCS4B03G0029800 and TraesCS5B03G1085500, were identified. In summary, this study establishes an efficient method for the measurement of cellulose crystallinity in wheat stems and provides a genetic basis for enhancing lodging resistance in wheat. [ABSTRACT FROM AUTHOR]