The root system， as the important 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 3B chromosome. The gene comprises seven exons and six 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 （pTaLAC69^-10In） and Hap2 （pTaLAC69^-10Del）. A dCAPS molecular marker was developed based on the InDel variation in the TaLAC69 promoter region， which can be used for the efficient identification of haplotypes in wheat. The average root length and the drought resistance coefficients of various indicators such as grain yield per plant， effective spike number per plant， main spike length and plant height of the wheat germplasm with haplotype Hap1 were significantly better than those of the germplasm with haplotype Hap2. Analysis of haplotype frequencies in 277 wheat germplasm population and 102 wheat population derived from Xiaobaimai revealed that the frequency of appearance of pTaLAC69^-10In showed a gradual decreasing trend with breeding years progressing from earlier to more recent. This study provides a key gene target and molecular marker for optimizing root architecture by using molecular biology techniques， which is conducive to accelerating drought resistance improvement in wheat.