Abstract: Serotype specific LUX (Light Upon Extension) real-time RT-PCR assays were developed for rapid detection of vesicular stomatitis virus (VSV). The LUX primers were designed based on the NS gene sequence of the NJ and IND serotype respectively. The real-time PCR assay could differentiate VSV from FMDV and other related RNA viruses. The detection limit of the assay could reach 1 TCID50 as showed by tests on serial ten fold dilution of cell propagated virus samples. Comparison tests indicated that the LUX real-time RT-PCR assay had at least ten-fold increase of sensitivity than the gel-based RT-PCR method. The assay could detect VSV infection and identify the serotype of the virus from import bovine clinical samples and artificial infected guineapig tissues. The whole procedure, including RNA extraction, realtime amplification and dissociation analysis, could complete within 3 hours.