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Waterlogging stress is one of the major abiotic stresses affecting the productivity and quality of many crops worldwide. However, the mechanisms of waterlogging tolerance are still elusive in barley. In this study, we identify key differentially expressed genes (DEGs) and differential metabolites (DM) that mediate distinct waterlogging tolerance strategies in leaf and root of two barley varieties with contrasting waterlogging tolerance under different waterlogging treatments. Transcriptome profiling revealed that the response of roots was more distinct than that of leaves in both varieties, in which the number of downregulated genes in roots was 7.41-fold higher than that in leaves of waterlogging sensitive variety after 72 h of waterlogging stress. We also found the number of waterlogging stress-induced upregulated DEGs in the waterlogging tolerant variety was higher than that of the waterlogging sensitive variety in both leaves and roots in 1 h and 72 h treatment. This suggested the waterlogging tolerant variety may respond more quickly to waterlogging stress. Meanwhile, phenylpropanoid biosynthesis pathway was identified to play critical roles in waterlogging tolerant variety by improving cell wall biogenesis and peroxidase activity through DEGs such as Peroxidase (PERs) and Cinnamoyl-CoA reductases (CCRs) to improve resistance to waterlogging. Based on metabolomic and transcriptomic analysis, we found the waterlogging tolerant variety can better alleviate the energy deficiency via higher sugar content, reduced lactate accumulation, and improved ethanol fermentation activity compared to the waterlogging sensitive variety. In summary, our results provide waterlogging tolerance strategies in barley to guide the development of elite genetic resources towards waterlogging-tolerant crop varieties. [ABSTRACT FROM AUTHOR]