Spot blotch of barley (Hordeum vulgare L.), caused by Bipolaris sorokiniana, is responsible for major losses in crop yield. Breeding-resistant barley varieties have proven to be an effective countermeasure for protecting agricultural production. Plants react to pathogen attacks by up-regulating secondary metabolites. Marker compounds for a B. sorokiniana infection are examined by untargeted UPLC-TOF-MS metabolomics and lipidomics techniques. Through the analysis of nine quantitatively resistant and susceptible barley genotypes, derived from the nested association mapping population HEB-25, followed by structure identification experiments and spore germination assays, 57 metabolites are identified. In addition to previously known metabolites, the unknown compounds 5-carboxydidehydroblumenol C-9-O-ß-d-glucoside (46) and grasshopper ketone 3-sulfate (47) were elucidated. 5-Carboxyblumenol C-9-O-ß-d-glucoside (45) was described for the first time in barley leaves. Pheophytin derivatives, oxylipins, linolenate-conjugated lipids, and flavone glycosides were described for the first time in connection with infections by phytopathogenic fungi or resistance in barley.