Wheat is a staple food crop and plays a critical role in global food security. The root system, as the core organ for water and nutrient uptake, exhibits a close association between its morphology and both yield and abiotic stress resistance.? This study cloned a root length regulating gene TaLAC69 from a GWAS-identified locus on chromosome 3B. The gene comprises 7 exons and 6 introns, encoding 551 amino acids with three conserved copper oxidase domains. Its promoter contains multiple hormone-responsive and abiotic stress-responsive cis-elements. The qRT-PCR analysis showed that its expression was significantly induced by abscisic acid (ABA), methyl jasmonate (MeJA), drought, heat and cold stress. The root length of the TaLAC69 knockout mutant seedlings was significantly shortened. One 3-bp insertion/deletion (InDel) variation in the promoter region and five single-nucleotide-polymorphisms (SNPs) in the coding region of TaLAC69 were identified, forming two haplotypes, Hap1 and Hap2. A dCAPS molecular marker was developed based on the InDel at the ?10 locus in the TaLAC69 promoter region, which can be used for the efficient identification of haplotypes in wheat. The germplasm with haplotype Hap1 (pTaLAC69_-10In⁾ exhibited significantly superior performance compared to the germplasm with haplotype Hap2 (pTaLAC69_-10Del⁾ in average root length, drought resistance coefficient of grain yield per plant (GYP-DRC), drought resistance coefficient of effective spikes number per plant (ESNP-DRC), drought resistance coefficient of main spike length (SL-DRC), and drought resistance coefficient of plant height (PH-DRC). Analysis of haplotype frequencies in 277 wheat germplasm population and 102 wheat population derived from Xiaobaimai revealed that pTaLAC69_-10In showed a gradual loss trend with the advancement of breeding years. This study provides a key gene target and molecular marker for optimizing root architecture via molecular breeding, which is conducive to accelerating drought resistance improvement in wheat.