Virulence spectra for 14 isolates of the barley powdery mildew pathogen considered to be added to the pathogen genebank were determined. The isolates were obtained from a sample of aerial pathogen population collected across the Czech Republic in 2009. The spectra were determined on 28 barley varieties, possessing mostly specific resistance genes and inoculated with the isolates examined. Based on differentiation among each other, all 14 isolates showed their originality. None of the isolates exhibited increased pathogenicity to a variety with the non-specific resistance gene mlo9. All isolates were virulent on varieties carrying specific resistance genes Mla8 and Mlra, and in contrast, none of the isolates was virulent on a variety with the gene Mla23 and with a combination of genes Mla3, MlaTu2. Required characters, such as virulence on the resistance Lv or avirulence on the gene Mlh, were confirmed in some isolates. The results represent a significant step toward obtaining a complete set of information about every isolate before its including in the working genebank of the pathogen.