After removing gliadin and other proteins with 7.5% 2-propanol and 0.3 mol/L NaI, the total glutenin subunits were extracted with two kinds of buffer, one containing 25% 2-propanol, 0.04 mol/L Tris-HCI (pH=8.0), 10% SDS and 2% DDT and the other containing 25% 2-propanol, 0.04 mol/L Tris-HCI (pH=8.0), 10% SDS and 1.4% VP. In SDS-PAGE electrophoresis (4% stacking gel and 13% resolving ge1) system HMW-GS and LMW-GS were clearly separated by the improved method with only a single step. The background of the SDS-PAGE gel was gliadin-free. It was more effective and applicable to separate and distinguish the complex LMW-GS. This method will be used in wheat protein separation, variety identification and improvement.