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Not AvailableLate leaf spot (LLS) caused by fungi Passalorapersonatais generally more destructive and difficult tocontrol than early leaf spot. The aim of this study was todecipher biochemical defense mechanism in groundnutgenotypes againstP. personataby identifying resistancespecific biomarkers and metabolic pathways induced duringhost–pathogen interaction. Metabolomics of non-infectedand infected leaves of moderately resistant (GPBD4 andICGV86590), resistant (KDG128 and RHRG06083) andsusceptible (GG20, JL24 and TMV2) genotypes was carriedout at 5 days after infection (65 days after sowing). Non-targeted metabolite analysis using GC–MS revealed total 77metabolites including carbohydrates, sugar alcohols, aminoacids, fatty acids, polyamines, phenolics, terpenes and ster-ols. Variable importance in projection (VIP) measure ofpartial least squares-discriminant analysis (PLS-DA)showed that resistant and moderately resistant genotypespossessed higher intensities of ribonic acid, cinnamic acid,malic acid, squalene, xylulose, galactose, fructose, glucose,b-amyrin and hydroquinone while susceptible genotypes hadhigher amount of gluconic acid 2-methoxime, ribo-hexose-3-ulose and gluconic acid. Heat map analysis showed thatresistant genotypes had higher intensities ofb-amyrin,hydroquinone in non-infected and malic acid, squalene,putrescine and 2,3,4-trihydroxybutyric acid in infectedleaves. Dendrogram analysis further separated resistantgenotypes in the same cluster along with infected moderatelyresistant genotypes. The most significant pathways identifiedare: linoleic acid metabolism, flavone and flavonol biosyn-thesis, cutin, suberin and wax biosynthesis, pentose andglucuronate interconversions, starch and sucrose metabo-lism, stilbenoid biosynthesis and ascorbate and aldaratemetabolism. Targeted metabolite analysis further confirmedthat resistant genotypes possessed higher content of primarymetabolites sucrose, glucose, fructose, malic acid and citricacid. Moreover, resistant genotypes possessed higher con-tent of salicylic, coumaric, ferulic, cinnamic, gallic acid(phenolic acids) and kaempferol, quercetin and catechin(flavonols). Thus metabolites having higher accumulation inresistant genotypes can be used as biomarkers for screeningof LSS resistant germplasm. These results unravel thathigher amount of primary metabolites leads to stimulate theaccumulation of more amounts of secondary metabolitessuch as phenolic acid, flavanols, stilbenes and terpenoids(squalene andb-amyrin) biosynthesis which are ultimatelyinvolved in defense mechanism against LLS pathogen.Not Availabl