European Union regulations provide important guidelines for maintaining the Protected Designation of Origin (PDO) of olive oil and other foods. This includes characterization of foods based on variety (cultivar) and geographical origin, as this may be used as a criterion for determining authenticity and quality. Therefore, analytical method standards need to be established to ensure these criteria. This study describes how cultivar differences can be established between Italian oils, obtained from single varieties, based on acid, sterol, and TAG differences determined by chemometrics. TAG and FA composition provided the best basis for differentiation of olive oils among cultivars. The results were compared with those obtained using C-13 NMR analysis, and a similar differentiation between oils of different cultivars was achieved. C-13 NMR provides useful information on the acyl composition and on the positional distribution of the glycerol moiety and can be used for classification of cultivars based on oil composition. Furthermore, the advantages of this technique come from the rapidity with which information can be obtained and from the very simple preparation procedure required for analysis.