In this study, we carried out the first large-scale phosphoproteome analysis of seedling leaves in Brachypodium accession Bd21 using TiO2 microcolumns combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MaxQuant software. A total, 1,470 phosphorylation sites in 950 phosphoproteins were identified. Of the 950 phosphoproteins identified, 127 contained 3 to 8 phosphorylation sites. The phosphoproteins and phosphosites identified in our study expanded our knowledge of protein phosphorylation modification in plants, especially in monocot.