Drought stress is a major abiotic stress affecting plant growth and development. Brachypodiumdistachyon L. is a special model plant for wheat, barley, and several potential biofuel grasses. In this study, we make comparative phosphoproteome analysis of seeding leaves at the three leaf stage in Brachypodiumdistachyon L. for the different treatment conditions (6h, 24h). A total of 480 unique phosphopeptides, representing 439 phosphoproteins, were identified under drought stress. Motif-X analysis identified six motifs, such as [SP], [RxxS], [LxRxxS], [SxD], [SF] and [TP]. Molecular function and PPI analysis suggested that many proteins may take participation in signal transduction, gene expression, drought response and defense with energy metabolism and material transmembrane transport under moisture deficiency condition. Further analyses revealed that these SCPL phosphoproteins may play important roles in regulating signal transduction and material transmembrane transport to cope with the adverse environmental conditions. This study first focus on the effects of phosphorylation modification mechanisms on the growth and development of seeding leaves at different drought stresses.