Wheat grain protein content (GPC) is an important quality indicator. The GPC of wheat grown in the middle and lower reaches of the Yangtze River is often low. Marker-assisted selection (MAS) is an effective tool for improving quantitative traits; however, most markers have not been effectively applied in GPC improvement except Gpc-B1, although many loci associated with GPC were identified. In this study, linkage analysis using a recombinant inbred line population from the cross of core parents of Ningmai 9 and Yangmai 158 and association mapping using the local cultivated varieties were performed and nine candidate intervals were identified. The appropriate kompetitive allele specific PCR (KASP) markers associated with GPC were successfully developed and screened in 1,163 F4 breeding lines. Three markers, Kgpc-2B, Kgpc-2D, and Kgpc-4A, were validated to be significantly related to GPC by large-scale association mapping, and they were combined to achieve the highest efficiency to enhance GPC. We applied these markers in 164 F6 breeding lines and obtained 15 lines with high GPC, indicating their high selective efficiency. Further, strategies for gene exploration in the three significant intervals were proposed. These results were expected to provide a novel route for improving GPC in wheat quality breeding.