A novel QTL, TaqW-6B of water-extractable arabinoxylan content in the wheat grain on chromosome 6BL was identified and fine mapped in a narrow region 3.8Mb. Water-extractable arabinoxylan (WE-AX), an important component of hemicellulose, is associated with various abundant health benefits. In this study, QTLs for WE-AX content were detected in two populations: (1) a recombinant inbred line (RIL) population with 164 lines derived from a cross between Avocet and Chilero (AC population) genotyped with diversity array technology (DArT), and (2) a natural population of 243 varieties (CH population) genotyped with the Axiom wheat 660K single-nucleotide polymorphism (SNP) array. A stable QTL Qwe-ax.haust-6B, explaining 8.51-15.59% of the phenotypic variance, was mapped in the physical interval 459.38-572.09Mb on the long arm of chromosome 6B in the AC population, tightly linked with DArT markers 3,944,740 and 4,991,038 under three experimental conditions. The Qwe-ax.haust-6B was further narrowed down to be delimited in the physical interval 516.47-571.58Mb on chromosome 6BL, explaining 5.86-16.27% of the phenotypic variance in the CH population. Furthermore, we developed high-throughput kompetitive allele-specific PCR (KASP) markers to reconstruct the genetic linkage map in the AC population, and Qwe-ax.haust-6B was fine mapped into a narrow region named TaqW-6B, which was compressed between KASP-6B-3 and KASP-6B-6 at a physical distance of 3.8Mb. In the meanwhile, the markers were also validated in a natural population of 160 wheat lines (NP population). Consequently, this study is of great importance to provide the theoretical basis for cloning the key gene and developing functional markers for molecular breeding.