The biotrophic [Blumeria graminis (DC.) Speer] and necrotrophic [Cochliobolus sativus (S. Ito & Kurib.) Drechsler ex Dastur] are economically important pathogens of barley found worldwide. To better understand the defense responses initiated by resistant barley challenged with B. graminis and C. sativus, important known defense-related genes at early points of infection were analyzed using PCR (qPCR) approach. Time-course experiments revealed a remarkable discrepancy in the expression patterns of the defense-related genes between barley B. graminis or C. sativus interactions, and all of them exhibited significant differential accumulations compared to the non-inoculated controls. The most outstanding differences were observed in PR1 and PR5 expression which were 6 and 9 folds higher respectively at the 72-hpi of barley B. graminis interaction as compared with C. sativus. Data showed that barley plants triggered different defense mechanisms to strengthen their B. graminis and C. sativus resistance, and the expression patterns of the same defense-associated genes were altered in adaptation to each pathogen. Taken together, our study might provide useful information for a deeper molecular research on barley defense responses against pathogens with different lifestyles.