Key message <p>Metabolomics and transcriptome profiling were used to identify drought-responsive metabolites and genes of two sorghum cultivars. Respiratory metabolism may be closely associated with the enhanced stress resistance of sorghum. Our results provide a valuable resource for the breeding of new germplasms with strong drought tolerance.</p> Abstract <p>Suitable drought stress can enhance the accumulation of specific metabolites in plants. Sorghum, a drought-tolerant grain crop, can be used as the optimal material to analyze its drought tolerance mechanisms. The purpose of this study is to explore the metabolic and transcriptional regulatory mechanisms of sorghum in response to drought stress. In this study, physiological factors, metabolome, and transcriptome of sorghum cultivars with contrasting drought resistance (SCS, Sanchisan; JL, Jinliang 5) were analyzed upon drought stress. The results showed that under drought stress, soluble sugar contents in SCS leaves significantly increased by 112.2% and betaine and POD levels in JL leaves also significantly increased. According to metabolome analysis, a total of 374 differentially accumulated metabolites (DAMs) were obtained. For example, the content of 3-hydroxy-3-methylbutanoic acid in SCS increased by over 16-fold and that of 1,2-dipalmitoylphosphatidylglycerol in JL increased by over fourfold under drought stress. In addition, a total of 7031 differentially expressed genes (DEGs) were obtained, and there were 306 common DEGs between SCS and JL, 6229 unique to SCS, while 496 unique to JL. In the KEGG analysis, DEGs in both SCS and JL were significantly enriched in “plant hormone signaling”. Chloroplastic beta-amylase 1 (EC3.2.1.2) gene <i>LOC8070506</i> and alpha-glucosidase (EC3.2.1.20) gene <i>LOC8061607</i> levels increased in both cultivars under drought conditions, and their levels were higher in SCS than in JL. These findings enhance our understanding of the molecular mechanism of drought tolerance in sorghum.</p>