Using PCR-denaturing gradient gel electrophoresis (DGGE) targeting the RNA polymerase beta subunit (rpoB) gene, a simultaneous detection method for Vibrio species was established. rpoB gene-based PCR-DGGE was carried out with eight Vibrio Reference strains (each from different species), mixed sample (including these Vibrio Reference strains),two non Vibrio strains, four environmental Vibrio strains, and three unidentified environmental strains. For comparison, 16S rRNA gene-based PCR-DGGE of the eight Vibrio Reference strains was performed with universal primers. In addition, three unidentified strains were identified by 16S rRNA and gyrB gene sequencing and API20E system in order to confirm the accuracy of rpoB gene-based PCR-DGGE detection. Results revealed that rpoB-based PCR-DGGE could well discriminate eight Vibrio Reference strains and could not discriminate different strains within the same species. The bands derived from two non Vibrio strains could not match with any bands in Reference marker. Meanwhile, 16S rRNA gene-based DGGE failed to distinguish these Reference strains. Furthermore, four out of eight Vibrio species exhibited heterogenous bands in 16S rRNA gene-based DGGE. Sequencing and API 20E identification of unidentified strains coincided with the detection by rpoB gene-based PCR-DGGE. The results demonstrated that rpoB-based PCR-DGGE provided a rapid and efficient method for simultaneous detection of multiple Vibrio species, which can avoid the limitations inherent in 16S rRNA gene-based PCR-DGGE.