Presymptomatic and accurate diagnosis of Mycosphaerella graminicola leaf blotch is desirable for the disease prediction and the timely application of fungicides. To develop a sensitive PCR assay, four specific primer pairs were designed. They were more specific than three known specific primer pairs. Three of them could detect as little as 0.5 pg M. graminicola DNA in a conventional PCR. A real-time PCR assay was applied for monitoring the disease progression in both inoculated and naturally infected wheat plants using the primer pair ST-rRNA F/R. In inoculated plants, M. graminicola DNA could be detected immediately after inoculation and a steady increase was detected before visible symptoms appeared at 8 days. The rapid growth period took place between 6 and 16 days postinoculation. In the field, the disease progression in the top three leaf layers was followed during the epidemic period. The results were significantly correlated to the disease indices (R=0.8986) and also to the number of pycnidia per leaf (R=0.9227). These suggest that the real-time PCR assay is a reliable approach for the presymptomatic and accurate detection of M. graminicola development in the field.