Lysine 2-hydroxyisobutyrylation (Khib) is a novel naturally occurring post-translational modification. The system Khib identification at proteomics level has been performed in various species and tissues to characterize the role of Khib in biological activities. However, the study of Khib in plant species is relatively less. In the present study, the first plant root tissues lysine 2-hydroxyisobutyrylome analysis was performed in wheat with antibody immunoprecipitation affinity, high resolution mass spectrometry-based proteomics and bioinformatics analysis. In total, 6328 Khib sites in 2186 proteins were repeatedly identified in three replicates. These Khib proteins showed a wide subcellular location distribution. Function and pathways characterization of these Khib proteins indicated that many cellular functions and metabolism pathways were potentially affected by this modification. Protein and amino acid metabolism related process may be regulated by Khib, especially ribosome activities and proteins biosynthesis process. Carbohydrate metabolism and energy production related processes including glycolysis/gluconeogenesis, TCA cycle and oxidative phosphorylation pathways were also affected by Khib modification. Besides, root sulfur assimilation and transformation related enzymes exhibited Khib modification. Our work illustrated the potential regulation role of Khib in wheat root physiology and biology, which could be used as a useful reference for Khib study in plant root.