Barley (Hordeum vulgare L.) is a major crop in Australia and powdery mildew (Blumeria graminis f. sp. hordei) is one of its most common diseases. Genes for resistance to powdery mildew were postulated for 86 Australian barley varieties and nine advanced breeding lines using 40 reference isolates of the pathogen. Fifty isolates collected in Australia in 2011 were used for additional tests of some varieties. In total, 22 known resistance genes [mlo, Mla1, MlaAl2, Mla3, Mla6, Mla7, Mla8, Mla9, Mla12, Mla13, Mlat, Mlg, MlGa, Mlk1, MlLa, Mlra, Ml(Ab), Ml(Ch), Ml(Dr2), Ml(He2), Ml(Lo) and Ml(St)] were detected. The most frequent genes were Mla8 and Mlg present in 43 and 34 varieties, respectively, while MlGa was found in 12 varieties. Each of the specific resistance genes Mla1, Mla3, Mla6, Mla9, Mla13, Ml(St) and the non-specific recessive gene mlo was found in one variety only. The varieties Maritime and Stirling appear to carry no specific resistance genes. Fifteen unknown resistances were detected. It is recommended that Australian barley breeding programs exploit European varieties possessing mlo to improve the resistance to powdery mildew in new varieties.