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In order to evaluate the efficiency of inter-simple sequence repeat (ISSR) markers for Olea europaea L. varietal identification purposes, ten primers constituted by simple sequence repeats were tested against drupes and leaves from ten Italian O. europaea cultivars. Leaf DNA was initially used to assess optimal PCR conditions as well as to screen the effectiveness of the primers. Subsequently, DNA extraction from drupes, rich in PCR interfering substances such as phenolic compounds and lipids, was set up. An analysis of primer informativeness in the chosen set of cultivars was also carried out. By combining the amplification patterns from two primers, namely UBC-841 and UBC-808, that were the most polymorphic in terms of both polymorphic/amplified bands ratio and resolving power, it was possible to distinguish the drupes from all the examined cultivars.