Abstract: For a rapid detection of Batai virus (BATV) in ducks, a one-step reverse transcription polymerase chain reaction (RT-PCR) assay was developed based on the conserved region of the M gene in BATV. With the optimized reaction conditions, this assay could specifically amplify a 480 bp fragment from the M gene. There were no similar gene amplifications on 5 other pathogens found in ducks, such as avian Tembusu virus and duck hepatitis virus. The testing sensitivity of the method was 1×10^1.1 TCID₅₀ with a high reproducibility. Consequently, this newly developed methodology seemed to be highly specific and sensitive for BATV detection, and could be applied for rapid and early diagnosis as well as epidemiological investigations on BATV.