Morphological and molecular characterization for six seedling genotypes of Pistachio vera L (V1,V2,V3,V4,V5, and V6) in comparison with the most commercial and wide spread varieties, Ashoury andWhite batoury was carried out in Swaida province, southern of Syria. Nuts chemical contents (dry substance, moisture, total sugar and oil content) for both fresh and dry nuts weight were also evaluated. Seedling genotypes showed obvious differences toward the main diagnostic traits, such as alternate bearing phenomena, the ratio of kernel weight to total nut weight, split nuts percentage and loss percentage. Genotype V4 was recognized by some important characters compared with the other genotypes studied. Cluster analysis was assessed according to the existence or absence of the most important studying morphological and agronomic indexes. All genotypes studied and comparative cultivars were clustered into four groups. RAPD technique was achieved using 25 randomly primers, 19 of which were polymorphic with an average of polymorphism percentage of (66.47%). The highest value of genetic similarity (0.81) was between Ashoury and V5 genotype, whereas the lowest value (0.56) was between V3 and V6. Cluster analysis depending on RAPD data divided the population studied into two main groups. RAPD unique band was estimated (37 unique band, 21 positive and 16 negative). It was concluded that RAPD technique can be used efficiently to emphasize, identify, insure and evaluate the genetic diversity of Pistachio vera.