The mechanism of infection and the latency period of caterpillar fungi, such as Isaria sinclairii Kobayasi largely remain unclear. In this study, we tested the reliability and suitability of specific primer set (PC-F/ PC-R) using PCR analysis to detect I. sinclairii in the forest soil. Soil samples were taken from the sites with fruiting initiation of I. sinclairii. Total DNA was extracted from soil samples and analyzed with respect to the collecting phase and soil depth. The mycelia of I. sinclairii were detected abundantly in the month of August after fruiting body development than any of October to December month. In addition, the mycelia of I. sinclairii were also detected from a depth ranging 5 to 30 cm. The method developed in this study offers effective tool for the elucidation of the biological characteristic of caterpillar fungi.