Barley quality depends on its molecular composition, particularly the amount, identity, and structures of the starch and protein. We assessed protein composition changes in response to short periods of heat stress under field conditions and in temperature-controlled glasshouses for two barley malting varieties that have similar phenology but different genetic backgrounds: RGT Planet and LaTrobe. Heat stress (>40˚C for 5 or 7 days) was imposed on selected plants when the individual tillers were at development stages ranging from anthesis to early grain fill. In the field, tillers were harvested weekly commencing immediately after the period of heat stress was applied, until maturity. In the glasshouse trial, tillers were harvested each day during the period of heat stress and again at maturity. Grain samples were analysed to assess the composition of the accumulating proteins with SWATH/DIA-MS proteomics. Peptides were compared to a database of known barley proteins and the confidently-identified proteins were compared across treatments. Major differences were observed in the protein response patterns of the two genotypes, in response to heat stress. A greater understanding of change to protein composition following heat stress will help improve understanding of the impacts on barley malting and brewing quality.