UNLABELLED Filterability is an essential quality parameter of barley malt and significantly impacts productive efficiency and quality of beer. In the study, differences of metabolic capability, rather than of initial contents of macromolecules in barleys, were found to be the main reason for malt filterability gap between the widely used cultivars Dan'er and Metcalfe in China. Comparative proteomics based on fluorescent difference gel electrophoresis (DIGE) was employed to quantitatively analyze proteins of four commercial malts belonging to the two cultivars, and 51 cultivar-differential spots were identified to 40 metabolic proteins by MALDI-TOF/TOF mass spectrometry, mainly including hydrolases and pathogen-related proteins. According to their function analysis and abundance comparison between cultivars, filterability-beneficial and -adverse proteins were putatively proposed. Two most remarkable differential proteins, β-amylase and serpin Z7, were further investigated to verify their effects on Dan'er malt filterability. These results provide biological markers for barley breeders and maltsters to improve malt filterability. BIOLOGICAL SIGNIFICANCE To the best of our knowledge, this is the first report of comprehensive investigation of metabolic proteins related to wort filterability of barley malts, and sheds light on clues for filterability improvement. Visible differences in the expression level of metabolic proteins between Dan'er and Metcalfe malts using 2D-DIGE signify a valuable tool for cultivar comparison, illustration of key proteins responsible for filterability and even other qualities of barley malts. And with these explorations on biomarkers of malt filterability and other aspects, there will be higher efficiency and quality of beer brewing, less application of exogenous hydrolases and more expending market for Chinese malting barleys. This article is part of a Special Issue entitled: Translational Plant Proteomics.