Two loci inhibiting Fhb1 resistance to Fusarium head blight were identified through genome-wide association mapping and validated in biparental populations. Fhb1 confers Fusarium head blight (FHB) resistance by limiting fungal spread within spikes in wheat (type II resistance). However, not all lines with Fhb1 display the expected resistance. To identify genetic factors regulating Fhb1 effect, a genome-wide association study for type II resistance was first performed with 72 Fhb1-carrying lines using the Illumina 90K iSelect SNP chip. Of 84 significant marker-trait associations detected, more than half were repeatedly detected in at least two environments, with the SNPs distributed in one region on chromosome 5B and one on chromosome 6A. This result was validated in a collection of 111 lines with Fhb1 and 301 lines without Fhb1. We found that these two loci caused significant resistance variations solely among lines with Fhb1 by compromising the resistance. In1, the inhibitory gene on chromosome 5B, was in close linkage with Xwgrb3860 in a recombinant inbred line population derived from Nanda2419 × Wangshuibai and a double haploid (DH) population derived from R-43 (Fhb1 near isogenic line) × Biansui7 (with Fhb1 and In1); and In2, the inhibitory gene on chromosome 6A, was mapped to the Xwgrb4113-Xwgrb4034 interval using a DH population derived from R-43 × PH8901 (with Fhb1 and In2). In1 and In2 are present in all wheat-growing areas worldwide. Their frequencies in China's modern cultivars are high but have significantly decreased in comparison with landraces. These findings are of great significance for FHB resistance breeding using Fhb1.