AbstractTouriga Nacional is the most popular Portuguese grapevine cultivar. After twenty years of clonal selection, highly recommended qualitative and productive clones have been identified. In the genus Vitis several molecular markers have been used to characterize genetic diversity at the DNA level, namely microsatellites, ISSR (inter simple sequence repeat), AFLP (amplified fragment length polymorphism), and stilbene synthase–chalcone synthase (StSy–CHS) markers.In this work a novel pure DNA extraction methodology, which occludes the extract in an agarose matrix, thus allowing the removal of the remaining hydrophilic impurities, was used. An optimized and highly reproducible procedure for the detection of differences among Vitis vinifera L clones using the StSy–CHS gene family primer combination is described. With this methodology, 21 clones and a commercial cultivar, ‘Chardonnay’, were evaluated with three StSy–CHS markers. It was possible to obtain 14% discrimination within Touriga Nacional (TN) clones, identifying two different groups. The polyclonal origin of TN clones was previously verified with eight microsatellite markers. The methodology described could be used for clonal selection of grapevine cultivars. Copyright © 2004 Society of Chemical Industry