Fusarium head blight (FHB) of wheat and barley causes severe economic damage by reducing the grain yield and contaminating the grains with trichothecene mycotoxins. Most toxic trichothecene is the deoxynivalenol, which is also a virulence factor of the pathogen. FHB resistance is quantitative, and more than one hundred quantitative trait loci (QTLs) have been identified in wheat. However, resistance mechanisms governed by these QTLs are largely unknown. A non-target metabolomics was applied to identify the mechanisms of resistance governed by one of the major QTL Fhb1 in wheat derived from the cultivar Nyubai, using a high resolution LC-ESI-LTQ-orbitrap. Metabolites accumulated in the rachis and spikelets of wheat near isogenic lines with contrasting alleles of Fhb1 upon Fusarium graminearum or mock inoculation was compared. The resistance related metabolites were mapped on plant metabolic pathways. The resistance in Fhb1 was associated with salicylic acid and jasmonic acid signaling, which in turn, activated the deposition of cell wall appositions consisting of phenolic glucosides, flavonoids, lignans and hydroxycinnamic acid amides that are synthesized via the shunt phenylpropanoid pathway. Surprisingly, the Fhb1 derived from Nyubai was not associated with the conversion of deoxynivalenol (DON) to less toxic deoxynivalenol-3-O-glucoside (D3G) as in double haploid lines harboring Fhb1 from Sumai-3. The technology standardized here can be used to identify mechanisms of resistance in other QTLs identified in wheat and barley.