Wheat streak mosaic virus, the cause of wheat streak mosaic disease, is considered a potentially destructive disease of wheat (Triticum aestivum L.) in important wheat-producing countries. Despite its economic impact, little is known about the pathogenesis and wheat defense mechanisms against this virus. Understanding the biological mechanisms occurring during pathogenesis is essential for undeciphering physiological processes in wheat-WSMV interaction. To evaluate wheat resistance, we analyzed the metabolome of wheat resistant (Adl-cross) and susceptible (Marvdasht) cultivars against WSMV Marvdasht isolate at three time points 24, 48 and 72 hours post infection (hpi) using LC-ESI-QTOF. Collectively, 581 and 79 metabolites in both positive and negative ionization modes, were respectively used in multivariate analysis. The PLSDA model using first principal component of VIP (variable importance in projection) values combined with T-test (P˂0.0.05) to find differentially expressed metabolites. By calculation of fold change (FC) for each metabolites (FC (inoculated /mock) ˃1) and based on VIP˃2, metabolites related to resistance and plant defense were distinguished. At 24 and 48 hpi, the most important metabolites having higher FC in R cultivar were phenolic compounds such as coumarins, a limited number of lipids for example fatty alcohols and a number of unknown compounds, while at 72 hpi, in addition to coumarins and alkaloids, several amino acids were increased. Most of these metabolites in S cultivars were accumulated at later times, suggesting delayed activation of defense responses in S cultivars. On the other hand, after development of infection, a gradual increase in the FC of significant metabolites was observed especially at 48 hpi, but their amount decreased at 72 hpi. Interestingly, some resistance related metabolites that had dramatically higher FC in R compared to S cultivars at 24 hpi, showed slightly decreased levels in R cultivars at 48 and 72 hpi while they had higher FC in the next days in S cultivars. The strong activation may be correlated with the rapid blocking of the virus infection and plant resistance. For instance, fatty acids and lipids, which are fundamental reinforcements of all plant cells, not only provide structural integrity and energy for different metabolic processes, but also serve as precursors for some important signal transducers. This study reports the relationship between secondary metabolism and virus infection in R and S cultivars and provides new insights into aspects of plant-pathogen interaction and insight into mechanisms of resistance at the metabolite level.