Abstract: The properties and cultivars of malting barley are closely related with malt production and brewing process. Three commonly used Australia malting barley varieties ( Hindmarsh, Schooner and Vlamingh) , and domestic Supi#3 and #6 were investigated. Then standard two- dimensional electrophoresis profiles of hordeins from those five malting barley were obtained. Ten characteristic protein spots with significant differences of each malting barley cultivar and forty- three common protein spots of the three Australia barley maps were identified with matrix- assisted laser desorption / ionization time- of- flight ( MALDI- TOF/ TOF) , and seven characteristic proteins and thirty- four common proteins were successfully identified for constructing the protein marker library of the five malting barley cultivars. This research provided a basis to promote application of this technology in domestic brewery and malting companies.