Barley leaf stripe with the causal agent of Pyrenophora graminea is one of the most important barley diseases that considered a serious threat to this crop. During 2016, a sampling was conducted from cultivated barley at some regions of West Azerbaijan province (Miandoab, Naghadeh), East Azerbaijan province (Miyane, Maragheh), Ardebil province (Borjlo), and Zanjan province (Khorramdareh). Plant specimens infected washed for 10 minutes under a gentle tap water flow, then cut into smaller pieces (approximately 5 mm by 5 mm) with scalpel subjected to surface sterilization with 10% sodium hypochlorite solution for 1-2 min, rinsed three times by sterile water, one centimeter specimens were transferred to petri dishes containing 2% water-agar (WA) culture medium and 25 ° C temperature. Grown fungi were purified on 2% WA medium using single spore and 121 isolates of P. graminea were obtained. After morphological identification and molecular confirmation, pathogenicity evaluation of 20 selected isolates representatives of all regions and reaction of different cultivars of barley to the isolates were done by sandwich method. Seeds sterilized by 10% sodium hypochlorite for five minutes and placed in two fungal layers have grown on a PDA culture medium for contamination. Sandwiches containing isolates and seeds were kept at temperature of 14-12 °C and dark conditions for 20-15 days. The pathogenicity tests were done at 23 ± 2 °C and a relative humidity of 70-90% of greenhouse conditions. In order to assessment of pathogenicity severity of isolates on two sensitive and resistant cultivars, in total, three isolates with the highest pathogenicity were selected on Makoei (100%, 100% and 57.5%) and Fajr 30 (100%, 100% and 37.5%). Finally, three selected isolates were used to investigate the reaction of Valfajr, Turkaman, Bahman, Nimrooz, kavir, Dasht, Rihan and Nick cultivars. The results showed that Dasht and Bahman cultivars with lower pathogenicity were identified as resistant cultivars, and Nimrooz, Rihan and Nick cultivars were the highest level of symptoms and infection.