Olive (Olea europaea L.) tree and oil are signature figures of the Mediterranean culture. Because of its high economic value, olive oil is extremely vulnerable to fraud. The aim of this study was to develop molecular tests for authenticating cultivar and botanical origin in olive oils. In order to authenticate the botanical origin and detect adulteration, a plastid DNA region was utilized for standardizing a capillary-electrophoresis barcode assay. The performance of the assay was evaluated on series of olive oil : seed oil admixtures. The assay proved successful in identifying seed oils in olive oil down to a limit of 10%. The molecular assay described in this work enables adulteration detection regardless of compositional similarities between the adulterant and adulterated oil species, thus will complement the shortcomings of analytical chemistry approaches. In order to establish a DNA-based identification key to ascertain the cultivar origin of Turkish monovarietal olive oils, short fragments from five olive genes were sequenced for SNP (Single Nucleotide Polymorphism) identification. CAPS (Cleaved Amplified Polymorphic DNA) assays were designed for SNPs that alter restriction enzyme recognition motifs. When applied on the oils of 17 olive cultivars, a maximum of five CAPS assays were necessary to discriminate the varietal origin of the samples. Admixture detection threshold of the assays was identified as 20% when tested on olive oil admixtures. The SNP-based CAPS assays developed in this work can be used for testing and verification of the authenticity of Turkish monovarietal olive oils, for olive tree certification, and in germplasm characterization and preservation studies.