Objective Grey red?backed voles (Myodes rufocanus) are agile, fierce and hard to catch, thus, it is difficult to judge their gender by external appearance, especially for the juvenile voles. Therefore, it may cause difficulties to their allocation and later breeding in laboratories. The aim of this paper is to establish a rapid, simple and accurate method for gender identification of grey red?backed voles. Methods Fresh hair follicles were taken from 6 adult male voles, 3 adult females and 14 4?week?old juvenile voles, 5 male and 5 female 9?week?old Wistar rats, and 5 male and 3 female 6?week?old BALB/ c mice. The genomic DNA was extracted using Chelex?100 resin and the zinc?finger Y/ X gene (ZFY/ZFY) and the gene of sex?determining region of the Y (SRY) chromosome were amplified by PCR, and a double PCR amplification method was established. Results The ZFY/ZFY gene and SRY gene were simultaneously amplified from the male voles, while only the ZFY/ZFY gene was amplified from the females. The gender of all 23 voles, 10 Wistar rats and 8 BALB/ c mice were correctly identified with this method, and the PCR results were consistent with the phenotypic and autopsy results. Conclusions Using fresh hair follicles as experimental materials for gender identification of grey red?backed voles can alleviate shock and damage to the animals. The established double PCR amplification method is accurate,simple, rapid, and deserves to be used for gender identification of grey red?backed voles.