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Background: The Dirigent (DIR) gene family is pivotal for lignin polymerization and stress adaptation in plants, yet its systematic characterization in Sorghum bicolor (S. bicolor), a critical bioenergy crop, remains underexplored. Methods: Leveraging the S. bicolor genome database, we conducted a genome-wide identification, phylogenetic classification, and expression profiling of the DIR gene family. Evolutionary dynamics, gene structure variations, promoter cis-regulatory elements, and spatiotemporal transcriptome patterns were analyzed using bioinformatics and experimental validation (RT-qPCR). Results: A total of 53 SbDIR genes were systematically identified, exhibiting uneven chromosomal distribution. Phylogenetic analysis clustered them into five clades (DIR-a, DIR-b/d, DIR-c, DIR-e, DIR-f), with subfamily-specific exon number variations suggesting functional divergence. Evolutionary studies revealed tandem duplication (TD) as the primary driver of family expansion, accompanied by strong purifying selection. Promoter analysis highlighted abundant hormone- and stress-responsive cis-elements. Tissue-specific RNA-seq data revealed root-enriched expression of SbDIR2/4/18/39/44/53, implicating their roles in root development. Notably, SbDIR39 and SbDIR53 were significantly upregulated (2.8- and 5-fold, respectively) under 150 mM NaCl stress, underscoring their stress-responsive functions. Conclusions: This study provides the first comprehensive atlas of the DIR gene family in S. bicolor, elucidating its evolutionary mechanisms and tissue-specific/stress-induced expression profiles. Key candidates (SbDIR39/53) were identified as promising targets for molecular breeding or CRISPR-based editing to enhance stress resilience in S. bicolor. These findings lay a foundation for translating genomic insights into agronomic improvements. Full article