Α stable isotope ratio mass spectrometer was used for stable isotope ratio (i.e., δ13C, δ18O, and δ2H) measurements, achieving geographical discrimination using orthogonal projections to latent structures discriminant analysis. A total of 100 Greek monovarietal olive oil samples from three different olive cultivars (cv. Koroneiki, cv. Lianolia Kerkyras, and cv. Maurolia), derived from Central Greece and Peloponnese, were collected during the 2019–2020 harvest year aiming to investigate the effect of botanical and geographical origin on their discrimination through isotopic data. The selection of these samples was made from traditionally olive-growing areas in which no significant research has been done so far. Samples were discriminated mainly by olive cultivar and, partially, by geographical origin, which is congruent with other authors. Based on this model, correct recognition of 93.75% in the training samples and correct prediction of 100% in the test set were achieved. The overall correct classification of the model was 91%. The predictability based on the externally validated method of discrimination was good (Q2 (cum) = 0.681) and illustrated that δ18O and δ2H were the most important isotope markers for the discrimination of olive oil samples. The authenticity of olive oil based on the examined olive varieties can be determined using this technique.