Resistance of barley to Fusarium graminearum was studied using a pair each of resistant and susceptible black and yellow barley lines. The spikelets were inoculated with a trichothecene-producing isolate, a trichothecene-nonproducing isolate (tri5−), or a mock solution. Spikelets were collected 72 h after inoculation and metabolites were analysed using a LC-hybrid MS system. Metabolite abundances were used to identify the constitutive (RRC) and induced resistance-related metabolites (RRI). The pathogen virulence factor, DON, and its plant detoxification product, DON-3-O-glucoside (D3G), were also identified and designated as resistance-indicator (RI) metabolites. The RRC, RRI and RI metabolites were putatively identified. Jasmonic acid was significantly induced in barley following inoculation with a trichothecene-producing isolate, but not with a tri5− isolate. The former isolate reduced the induction of both the number and amount of RR metabolites. The metabolites cinnamic acid, sinapoyl alcohol, coniferin, catechin and naringin were identified only in response to the inoculation with a tri5− mutant. The abundances of p-coumaric acid, coniferaldehyde and sinapaldehyde increased more in response to the tri5− mutant than to the trichothecene-producing isolate. The total amount of DON synthesized and its conversion to D3G varied greatly between the resistant and susceptible black barley, but not in yellow barley. Interestingly, an increase in the amount of total DON produced was associated with a decrease in the conversion of DON to D3G. The roles of RRC, RRI and RI metabolites in plant defence and their further use as potential biomarkers in screening are discussed.