Powdery mildew, caused by the fungal pathogen Blumeria graminis (Bg) is one of the most important foliar diseases of barley globally. Barley plants have evolved complex signaling pathways during defense response against Bg, including plant hormones such as salicylic acid (SA) and pathogenesis-related proteins. Therefore, to better understand the defense mechanisms of resistant barley plants attacked by Bg, cooperative functioning between mla1, mla6, mla12, Rac1 genes and SA was evaluated at early time points of infection. mRNA was isolated 1, 2, 3, 4 and 6 days post inoculation and used for cDNA synthesis. The expression of mla and Rac1 genes was quantified using Quantitative reverse transcriptase PCR (qRT-PCR). SA measurement was performed by a high-performance liquid chromatography (HPLC) system. Data showed a remarkable increase in the expression patterns of mla and Rac1genes accompanied with an increase in SA level in infected plants 1 day post inoculation (dpi) when compared with non-infected plants. The most outstanding differences were observed in mla1 and Rac1 expressions which were 6.27 and 3.55 folds respectively higher, 4 dpi of barley Bg interaction, and were accompanied with an elevated SA level (610 ng/g). According to findings, this study might increase our understanding for a deeper molecular research on the cooperative functioning of mla & Rac1 genes and SA in resistant barley responses against Bg.