Powdery mildew is the most common disease of barley in the Czech Republic and winter barley plays a crucial role in the winter survival and reproduction of the pathogen. This study was aimed at identifying resistance genes in winter barley cultivars grown in the Czech Republic from 1971 to 2005 by using the method of postulation. Forty-one cultivars and a parental line were tested at the seedling stage for their reaction to 32 selected isolates of Blumeria graminis f. sp. hordei. In total, 26 resistance spectra were detected and the following 18 resistance genes were found: Mla6, Mla7, Mla8, Mla13, Mla14, MlaMu2, MlaNo3, MlaRu4, Mlg, Mlh, MlLa, Mlra, Ml(Bw), Ml(Ch), Ml(Dr2), Ml(Dt5), Ml(IM9) and Ml(St). Two cultivars ('Kiruna' and 'Sorna') exhibited heterogeneity for mildew resistance. Another source of Mla13 and a possible centre of origin of Ml(Bw) are discussed.