Key message: Metabolic profiling of Pratylenchus thornei resistant and susceptible wheat genotypes indicates that fatty acid, glycerolipid and flavonoid classes of metabolites constitutively expressed in resistant wheat roots could reduce nematode reproduction. Abstract: The root-lesion nematode Pratylenchus thornei reduces wheat production in many parts of the world. In this study the metabolic profiles of two wheat genotypes ‘QT16258’ (moderately resistant) and ‘Janz’ (susceptible) were compared at 8 weeks post inoculation with or without P. thornei. We performed untargeted liquid chromatography mass spectrometry analysis (LC–MS) of the wheat root samples. A total of 11,704 MS features were identified, out of which 765 MS features were annotated using in-house chemical standards. Principal components analysis (PCA) and partial least square discriminant analysis (PLS-DA) indicated dissimilarity of the metabolome between P. thornei resistant and susceptible genotypes. Two-way analysis of variance indicated that metabolic differences were mainly constitutive rather than induced by inoculation with P. thornei. Eighty-four annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘QT16258’ than ‘Janz’ and belonged to the following classes of metabolites: flavonoids, fatty acids, glycerolipids, alkaloids, tannins, nucleotides, steroid glycosides and terpenoids. Eighty-five annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘Janz’ than ‘QT16258’ and belonged to the following classes of metabolites: amino acids, sugars, flavonoids and alkaloids. Several metabolites at higher concentration in ‘QT16258’, including quercetin-3,4'-O-di-beta-glucoside (flavonoid), linoleic acid (fatty acid), lysophosphatidylethanolamine (glycerolipid), hirsutine (alkaloid), 1-methylsulfinylbutenyl-isothiocyanate (glucosinolate), could potentially strengthen the root cell walls to inhibit nematode penetration and/or reduce nematode motility. Some metabolites at higher concentrations in susceptible ‘Janz’, including phenolics, coniferyl alcohol and indole acetic acid conjugates, could be nematode attractants as well as part of a hypersensitive browning reaction to nematode invasion. © 2021, The Author(s), under exclusive licence to Springer Nature B.V.