Diketopiperazines (DKPs) are cyclic dipeptides which have been detected in a variety of natural products, especially in thermally treated or fermented foods and beverages, providing a metallic bitter taste. DKPs, mainly due to their characteristic heterocyclic system, have been reported to exhibit a broad spectrum of biological activities including antimicrobial, antiviral, antitumor, antihyperglycaemic and antimutagenic. In the present study, several DKPs were identified in seven different Greek varieties of processed olives using HR-LC-MSⁿ. The identification of DKPs in olive samples was achieved by comparison of their retention time and fragmentation pattern with reference DKP standards. The MSⁿ spectra were identical to confirm the presence of specific compounds because their results associate both fragmentation pattern and fragments’ intensity. Nine compounds were found out of a total of 19 standard DKPs. The most prominent diketopiperazine was the cyclo(Phe-Phe) followed by and cyclo(Phe-Pro). Varieties where most DKPs were identified were Kothreiki, Kalamon, Throumpoelies and Helidoni.