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Key Message: A novel major QTL for FHB resistance was mapped to a 6.8 Mb region on chromosome 2D in a Chinese wheat cultivar Ji5265, and diagnostic KASP markers were developed for detecting it in a worldwide wheat collection. Fusarium head blight (FHB) is a serious disease in wheat (Triticum aestivum L.) and causes significant reductions in grain yield and quality worldwide. Breeding for FHB resistance is the most effective strategy to minimize the losses caused by FHB; therefore, identification of major quantitative trait loci (QTLs) conferring FHB resistance and development of diagnostic markers for the QTLs are prerequisites for marker-assisted selection (MAS). Ji5265 is a Chinese wheat cultivar resistant to FHB in multiple environments. An F 6 population of 179 recombinant inbred lines (RILs) was developed from Ji5265 × Wheaton. The population was genotyped by genotyping-by-sequencing (GBS) and phenotyped for FHB Type II resistance in greenhouses. A major QTL, designated as QFhb-2DL, was mapped in a 6.8 Mb region between the markers GBS10238 and GBS12056 on the long arm of chromosome 2D in Ji5265 and explained ~ 30% of the phenotypic variation for FHB resistance. The effect of QFhb-2DL on FHB resistance was validated using near-isogenic lines (NILs) derived from residual heterozygotes from an F 6 RIL of Ji5265 × Wheaton. The two flanking markers were converted into Kompetitive allele-specific PCR (KASP) markers (KASP10238 and KASP12056) and validated to be diagnostic in a collection of 2,065 wheat accessions. These results indicate that QFhb-2DL is a novel major QTL for resistance to FHB spread within a spike (Type II) and the two KASP markers can be used for MAS to improve wheat FHB resistance in wheat breeding programs., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)