In this study high temperature (60°C) treatment was used to construct endophyte-free populations of Achnatherum sibiricum (L.) Keng. The results indicated that high temperature treatment was effective in killing the endophyte in the seeds. After 30 days’ treatment, seedlings were all uninfected. At the same time, high temperature treatment had no adverse effect on germination rate, germinating potential and germination index of treated seeds. On the contrary, high temperature improved initial root growth of the treated seedlings. Compared to other methods, high temperature treatment had some advantages such as cost low, procedure simple and environment friendly. Therefore, this method was an effective way to construct endophyte-free populations of Achnatherum sibiricum.