Abstract: The pathogen of Pythium aphanidermatum is a very important pathogen causing disease and yield losses on vegetables. Correct identification of pathogens lead to correct and effective control strategies and methods. However, the traditional methods based on morphological and physiological identification becomes difficult and time consuming because of the similarity amongst the different groups of species and intraspecific morphological variation frequently observed in different field isolates. Molecular techniques based on several DNA methods have been developed to identify Pythium spp. and also to understand the relationships between species. Morphology and physiology characteristics of Pythium aphanidermatum were investigated for identification and variability. The optimum pH levels of Pythium aphanidermatum grow in vitro were 7 and the optimum growing temperatures of the isolates recovery in this study was 30°C. The mycelial were thick and white with fluffy topography. Each isolate produced aseptate, hyaline mycelium, the oogonia terminal, globose and smooth. Antheridia mostly intercalary, sometimes, broadly sac shaped, monoclinous or diclinous, thick walled aplerotic oospores and lobed sporangia. Sequences of selected isolates obtained in this study were matched Pythium aphanidermatum (KY646468) from GenBank. The UPGMA dendrogram showed that there were variations within the population of Pythium aphanidermatum at similarity coefficient 0.1. These isolates obtained from diseased cucumber plants collected from different farms in the Jordan Valley in April 2017.