Wheat (Triticum aestivum L.) is one of the most important cereal crops worldwide, and its production is challenged by global climate change and a shortage of resources. The root system plays a vital role in uptaking water and nutrients and sensing soil environmental signals, and it has great potential to improve the final yield and stress tolerance of wheat. In order to further explore the genes regulating root development, this study focused on qRS-6D, located on chromosome 6D and spanning from 462,701,391 to 465,068,943, which was significantly associated with the total root length, root volume, root surface, and root fresh weight in our previous GWAS analysis. Firstly, its genetic effects were validated using an F₆ segregating population by comparing the root-related traits of homologous lines harboring the alternative haplotypes of this QTL. Then, the number of causal genes of this QTL was narrowed down to four with a transcriptome-wide association study. Additionally, qRS-6D has been demonstrated to have genetic effects on several yield- (kernel length, kernel width, and thousand-kernel weight) and plant structure-related traits (plant height, peduncle length, total tiller number, productive tiller number, flag leaf length, and flag leaf angle). Relatively, the frequency of the favorable haplotype increased with the wheat breeding practice. This study provides a reliable genetic locus to improve root development and structure and evaluate its application potential in wheat breeding improvement.