Identification of virulence factors in the population of barley powdery mildew, is a necessary step for development of barley cultivars resistant to the disease in different parts of a country. In the present investigation, trap nurseries consisted of Near Isogenic Lines (NILs) of barley powdery mildew, were grown in ten different regions of Iran under natural infection during 2000-2002. Data on disease severity and responses of NILs were taken according to Saari and Prescott (1975) method modified by Eyal et al. (1987). According to the results, the virulence frequencies of Blumeria graminis f.sp. hordei varied in different locations and years. Virulence on Mlk was most freguent in all locations, followed by Ml(La), Mla9 and Mlal8 respectively. Virulence on Ml-Spiti were observed only in Ahvaz and Dezful, and virulence on Mla17 were recorded with the lowest frequency in Minoodasht. Virulence on Mlk, Mla9 and Ml(La) appeared to be common in all regions. No virulence on Mla6, Mlg+MlCP, Mlp, Mla7+ MlAb, Mla3, Mla19, mlo, Mla13, Mla16, and Mla19 were detected.