[Objective] Understanding the genome size of Rhizoctonia cerealis is the basis for the genome sequencing and assembly. In this study, we estimated the genome size of R. cerealis via the quantitative real-time PCR method. [Methods] The partial translation elongation factor A gene (tef A) of R. cerealis strain R0301 was cloned and sequenced. Southern blot analysis indicated tef A was a single copy in the genome. Finally, we used the quantitative real-time PCR method to calculate the genome size of R. cerealis strain R0301 with the sequenced R. solani AG1-IA strain GD118 as the control. [Results] The quantitative real-time PCR method could accurately determine the genome size of R. solani and the genome size of R. cerealis R0301 was between 32.2-36.6 Mb. [Conclusion] Quantitative real-time PCR was a fast, highly accurate and reliable method for the genome size estimation of Rhizoctonia.