Background and Objectives Olive tree (Olea europaea L. ) is one of the most ancient tree crops which has been domesticated in the Middle East for about 6000 years. The romans enlarged its cultivation from the Greek islands throughout the Mediterranean basin, mainly along the African and European coasts as well as in the areas where large volumes of water have improved the climate. In the past, cultivar distinguishing approaches were based on morphological characteristics of leaf, fruit and stone. Recently, morphological traits with isozyme and DNA-based markers have been applied to the classification of cultivars. The aim of this study was the evaluation of morphological and molecular similarities between the new and the known cultivars of olive trees in the Fadak garden in Qom province in Iran. Materials and Methods In order to analyze genetic variations of olive tree genotypes in Qom province, the olive genotypes (Manzanilla, Sevilana, Mahzam, Arbequina, and Conservalia) and two superior cold resistant genotypes (Fadak and Tooba) were studied using morphological traits based on distinctness, uniformity and stability (DUS) guideline in the Fadak garden (Qom province, Iran) in 2013. In addition, a total genomic DNA was extracted from the leaves, and the PCR was carried out by a set of six inter simple sequence repeats (ISSR) markers for amplification. Then, amplification products from ISSR primer tests were characterized on 2% agarose gels immersed in 1X TBE buffer. The gels were stained with ethidium bromide. The PCR fragments were scored for the presence or absence as 1 and 0, respectively. Results In this study, analysis of variance was performed on 12 quantitative traits. Results showed there were significant differences (p < 0. 01, p < 0. 001) between all the traits and Fadak and Tooba separated from other genotypes by comparing the means of the tests (Duncan multiple range method). According to the biplot graph (the first two components) in the PCA method by quantitative traits, Arbequina, Manzanilla and Tooba were included in one group, and Fadak and Tooba were distinctly separated from other genotypes. Also, according to the biplot graph (the first two components) in the PCA method by qualitative traits, the distance between Fadak and Tooba was low. In the molecular analysis, all ISSR markers (six primers) showed polymorphism. The total band range was 8-21 (with 14. 66 as the average) and the PIC varied from 0. 231 to 0. 447 (0. 309 average). Based on the PCA method, Tooba was utterly separated from other genotypes. Discussion In this study, Fadak and Tooba were completely different from other varieties using morphological traits and ISSR molecular markers. Fadak was found to be similar to Sevilana and Conservalia. There was a high similarity between Fadak and Tooba. Also, no similarity was observed between the results of morphological and molecular traits.