A multiplex, real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that allowed simultaneous detection and rapid differentiation of vesicular stomatitis virus strains--New Jersey (VSV-NJ) and Indiana 1, 2, and 3 (VSV-IN1-3). This assay involves use of a set of VSV universal primers located in the L gene that amplify VSV-IN1-3 and VSV-NJ using probes that allow differentiation of the major serotypes Indiana and New Jersey. The assay was evaluated using reference VSV, foot-and-mouth disease virus, swine vesicular disease virus, and vesicular exanthema of swine virus. To estimate diagnostic sensitivity, 159 epithelial samples collected between 1996 and 2002 from naturally infected cattle in Colombia were used. The assay cut off was calculated by testing RNA extracted from 150 virus-negative bovine tissues consisting of tongue, soft palate, muzzle, coronary band, and lymph node. All infected cattle were test positive for VS by results of real-time RT-PCR analysis; results for 156 of 159 (98.1%) agreed with the serotype determination from the complement-fixation test. Amplification did not occur in any of the negative bovine epithelial samples, allowing the cut-off values for the assay to be set. The real-time RT-PCR assay was documented to be sensitive and specific for the detection of VSV-NJ and VSV-IN (1-3) strains from field samples in a single reaction, thereby supporting use of this assay in the differential diagnosis of vesicular virus diseases in cattle.