Grapevine (Vitis vinifera L.) is one of the most valued crops. Wine industry is pivotal to worldwide economy, with Portugal in the top ten producers. V.vinifera is prone to several diseases, being downy mildew, caused by Plasmopara viticola, a major threat. Every year fungicides are vastly applied with impact at environmental, economic and human health level. The main concern in today's agriculture is reducing phytochemicals use. To define new control strategies towards sustainability, identifying resistance biomarkers is vital. Previously, we highlighted the importance of lipid signaling events in grapevine resistance to P. viticola. We showed that linolenic acid, jasmonic acid precursor, is constitutively more accumulated in a resistant genotype. So as to access lipids as resistance biomarkers, we analysed the fatty acid (FA) content of different grapevine species and cultivars. Clear separation between resistant and susceptible accessions was observed. Total FA content was significantly higher in susceptible grapevine accessions. A higher linolenic to linoleic acids ratio was found in resistant genotypes, while Double Bond Index was higher in susceptible cultivars. Our results indicate a path leading to early screening for resistance biomarkers in grapevine breeding programs.