The study of the genetic relationships among Palestinian olive cultivars is very important in order to assess their genetic diversity and structural details. A collection of five cultivars including Roomi, Souri, Improved Nabali, Barenge K18 and Wild Type were analyzed by using the sequences of two genes' fragments in which a panel of twenty-five Single Nucleotide Polymorphisms (SNPs) were identified. It is found that this group of SNPs is not efficient enough to discriminate among all of the cultivars. However, it is useful for varietal survey and construction of a database of Palestinian olive cultivars and for providing additional information that could constitute the basis for a rational scheme of breeding programs.