Wheat is a staple cereal crop that is crucial for food security and human health. Improving wheat quality has become an essential task for allowing 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, and it was used to conduct a quantitative trait loci (QTLs) analysis for grain quality-related traits. Eight QTLs were identified for grain protein content (GPC), starch content, and wet gluten content in two environments. Among them, a major and environmentally stable QTL for GPC, qGPC4D, 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. In addition, 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 high-quality wheat breeding.