Wheat stripe rust, powdery mildew, and Fusarium head blight (FHB) are the three most important diseases in wheat worldwide. Growing resistant cultivars is the most economic and effective method to control these diseases. To assess the disease resistance of commercial wheat cultivars and regional trial wheat lines in the Huang-Huai-Hai region of China, 146 wheat entries were inoculated with the Chinese prevalent Puccinia striiformis f. sp. tritici (Pst) races CYR32, CYR33, CYR34, and Blumeria graminis f. sp. tritici (Bgt) isolate E09 under controlled greenhouse conditions, respectively; these entries were also tested with the mixed Pst races, Bgt and FHB isolates at adult-plant stage in the field, respectively. The results showed that 108 (73.97%), 83 (56.85%), 99 (67.81%), and 22 (15.07%) entries were resistant to CYR32, CYR33, CYR34, and E09 at the seedling stage, respectively; 102 (69.86%), 24 (16.44%), and 2 (1.37%) entries were resistant to stripe rust, powdery mildew, and Fusarium head blight at the adult-plant stage, respectively. Additionally, the possible resistance gene(s) in these entries were postulated by the closely linked markers of stripe rust resistance genes Yr5, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26, powdery mildew resistance gene Pm21, and Fusarium head blight resistance gene Fhb1. Combined with disease resistance and molecular markers tests, 62, nine, and three wheat entries were postulated to carry the Yr9, Yr17, Yr26 gene, respectively, and no entries contained Yr5, Yr10, Yr15, Yr18, Pm21, and Fhb1 gene. This study laid a theoretical foundation for rational utilization of these entries and gene in wheat breeding programs and disease control.