Proteins extracted from five soybean cultivars were separated by reversed-phase (RP) and size exclusion (SE) high performance liquid chromatography (HPLC). A RP-HPLC method was developed that permits protein separation of nondefatted flours from soybean cultivars. RP-HPLC showed that protein of these cultivars could be classified into two groups, one of which contained intermediate component peaks under identical conditions. Genetically closely related cultivars exhibited only small differences in their RP-HPLC chromatograms. Size exclusion high performance liquid chromatography (SE-HPLC) resolved extracts of nondefatted flours from various soybean cultivars into six common peaks which accounted for 80 to 94% of the total peak area. Soybean cultivars were primarily identified by the percent area of their fifth peak (fraction F5) which had the highest variability of the total peak area. Seed protein of the cultivars was closely related with the area percent of peak F2 (r = 0.91). SE-HPLC proved to be a rapid one step quantitative method with potential for assessing soybean cultivars on the basis of protein content.