In recent years, the incidence of wheat take-all disease is increasing in Huang-Huai area of China. To determine the virulence of Gaeumannomyces graminis isolates and to provide the scientific basis for screening resistant wheat cultivars, three inoculation and two investigation methods were compared in this study. The results suggested that the method with fungal-colonized agar plugs 2 cm below the wheat seeds in combination with the 0-6 disease grade standard was better than the others, which presented appropriate disease severity and less standard error. The virulence of 52 G. graminis isolates collected from Henan, Jiangsu, Anhui, and Shandong provinces in 2010 was then tested using the method mentioned above. The virulence was shown to be different significantly among the isolates tested. Finally, 69 wheat cultivars were tested for their resistance against G. graminis isolate G1037, which was aggressive during seedling stage. All the wheat cultivars tested were susceptible to G1037, and none of them was immune or highly resistant to G. graminis. However, Fanmai 5 and Taikong 6 were moderately resistant against G. graminis.