Wheat is a staple cereal crop that is crucial for food security and human health. Improving wheat quality has become an essential task for breeders to meet escalating market demand. In this study, a set of wheat-Aegilops tauschii introgression lines was developed from a cross between the high-yielding wheat variety Jimai 22 and Ae. tauschii Y215. A high-density genetic map containing 2,727 single nucleotide polymorphisms (SNPs) was constructed using a 55K SNP array to conduct quantitative trait loci (QTL) analysis for grain quality-related traits. Eight QTL were identified for grain protein content (GPC), starch content, and wet gluten content in the two environments. Among them, a major and environmentally stable QTL, qGPC4D, for GPC was identified, with favorable alleles contributed by Ae. tauschii Y215. Subsequently, qGPC4D was narrowed down to a 9.88 Mb physical interval through further fine mapping utilizing the introgression lines. Additionally, three linked SNP of qGPC4D were converted into high-throughput kompetitive allele-specific PCR (KASP) markers and validated in the introgression population. These findings offer promising candidate genes, elite introgression lines, and KASP markers for wheat high-quality breeding.