Bacterial pustule is a major bacterial disease in most soybean-growing regions of Korea, resulting in soybean yield reductions of up to 60%. In this study, we aimed to understand the differences in disease severity caused by three strains of Xanthomonas citri pv. glycines, including two newly differentiated strains (K29 and K100) and the conventional representative strain, 8ra. Seventeen major soybean cultivars and 14 breeding lines were inoculated and compared for their reactions to the three strains of Xcg. The two new strains caused higher levels of disease severity, resulting in a greater number of susceptible cultivars. The landrace PI 547711, like Williams 82, was highly resistant to all Xcg strains; however, the other six cultivars that were resistant to 8ra, including Daepung2, were susceptible to the new strains (K29 and K100). In particular, the most widely grown cultivars, Daewon and Daepung2, were highly susceptible to K100. All of the breeding lines except two were also vulnerable to the new strains, K100 and K29. Of the breeding lines, SS05004-1-1-4-2-3-4 and SS05022-5-4-2-4-4-4 were resistant to the three Xcg strains, at similar levels to that of Williams 82. The breeding lines and cultivars evaluated in this study may be used to develop breeding materials resistant to new Xcg strains in the future. In addition, establishment of an improved disease evaluation system for bacterial pustule, by using multiple representative strains, is strongly recommended for breeding programs, to increase the efficiency and accuracy of selection for resistance as the pathogenic diversity of X. citri pv. glycines changes.