The mechanisms of resistance in barley to fusarium head blight (FHB), caused by Gibberella zeae are complex. Metabolomics technology was explored to phenotype resistance. Spikelets of barley genotypes with contrasting levels of resistance to FHB, mock inoculated or with the pathogen, were extracted with aqueous methanol and the metabolites were analyzed using liquid chromatography and hybrid mass spectrometry. Peaks were de-convoluted using XCMS and annotated using CAMERA and IntelliXtract bioinformatics tools. A t-test, of a total of 1608 purified peaks, selected 626 metabolites with significant treatment effects, of which 161 were identified as resistance related (RR) metabolites. A total of 53 metabolites, that are RR or pathogenicity related (PR), were assigned with putative compound names. These mainly belonged to three metabolic pathways: fatty acid (jasmonic acid, methyl jasmonate, 9,10-dihydroisojasmonate, linolenic acid, linoleic acid, traumatic acid), phenylpropanoid (p-coumaric acid, caffeyl alcohol, dimethoxy-4-phenylcoumarin, rosmarinic acid, diphyllin, 5-methoxypodophyllotoxin) and flavonoid (naringenin, catechin, quercetin, and alpinumisoflavone). A few PR/RR metabolites significantly reduced mycelial growth of G. zeae in vitro.