Abstract: In this study, physiological and molecular changes caused by different concentrations of tetraconazole were investigated in barley. The analyzes were carried out 7 days after the application of tetraconazole to the seedlings. In physiological analysis, root and shoot length, water loss rate (WLR) and ion leakage were assessed. No significant differences (p > 0.05) were detected overall in tetraconazole treated plantlets. Similarly, increased concentrations of tetraconazole led to no scientifically significant differences on experiment sets. WRKY9, WRKY33 and WRKY34 genes expression showed no alteration in response to increased concentrations of tetraconazole. Tetraconazole also did not induce DNA alterations with 91.13% GTS and DNA methylation polymorphism ratios, with ranges from 5.7 to 6.8% based on RAPD and CRED-RA, respectively. This is the first report that tetraconazole has no negative effects on barley at the physiological and molecular level and seems to be safe and friendly fungicide.