Epidemic haemorrhagic disease virus (EHDV) is an arbovirus that seriously endangers ruminants. Currently,nine serotypes of EHDV have been discovered worldwide,and five of them are prevalent in China. Neutralization test is the gold standard for serotype identification of EHDV,which has the shortcomings of time-consuming,laborious and long experimental period. Therefore,establishing an accurate and rapid serotyping method for EHDV is of great significance for the prevention and control of this disease. In this study,the second gene segment (Seg-2),which determines the serotype of epizootic haemorrhagic disease virus (EHDV),was used as the target gene to establish a one-step RT-PCR method for serotype-specific detection of EHDV. The results of specificity test showed that the method could accurately identify different serotypes of EHDV,and had no cross-reaction with bluetongue virus,chuzan Virus and akabane virus. The results of sensitivity test showed that the detection limit of the EHDV nucleic acid for different serotypes could reach 102 copies. EHDV strains isolated from different times and different regions in China were identified by this one-step RT-PCR and the results showed that the 31 EHDV isolates belonged to five serotypes,inculding EHDV-1,-5,-6,-7 and -10. The results of the RT-PCR were completely consistent with the results of the serum neutralization test. The above results showed that the method established in this study had good specificity and high sensitivity,and could quickly and accurately identify the serotype of EHDV strains.