The qualitative characteristics and chemical parameters were determined for 112 virgin olive oil samples of the two dominant olive cultivars in the southern region of Peloponnese, cv. Koroneiki and cv. Mastoides. As no relevant data exist for this geographical area, yet one of the most important olive-growing regions in Greece, this study aimed to evaluate and evidence the differences on specific chemical characteristics of the oils because of their botanical origin. Olive oils of Koroneiki variety were characterized by a three-fold lower concentration in heptadecanoic and heptadecenoic acid compared to oils of cv. Mastoides. In addition, Mastoides oils exhibited higher β-sitosterol and total sterols concentration and lower Δ-5-avenasterol and total erythodiol content compared to Koroneiki olive oils Analysis of variance and principal component analysis of the GC-analyzed olive oil samples showed substantial compositional differences in the fatty acid and sterolic profile between Koroneiki and Mastoides cultivars. Hence, results demonstrate that the fatty acid and sterolic profile can be used as exceptional compositional marker for olive oil authenticity.