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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, <i>TraesCS4B03G0029800</i> and <i>TraesCS5B03G1085500,</i> 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.