Aims To explore a prokaryotic species-specific DNA marker, 16S-23S rRNA gene internal transcribed spacer (ITS) sequence for identification and classification ofVibrio. Methods and Results Five hundred and seventy four ITS sequences from 60Vibriostrains were collected, then the primary and secondary structures of ITS sequence were analysed. The ITS was divided into several subunits, and the species-specificity of these subunits were evaluated byblast. The variable subunit of ITS showed high species-specificity. A protocol to identify aVibriospecies based on ITS analysis was developed and verified. Both the specificity and sensitivity were 100%. The phylogeny analysis ofVibriobased on ITS showed that ITS devised a better classification than 16S rDNA. Finally, an identification method ofVibriobased on ITS sequencing in food samples was developed and evaluated. The results of ITS sequencing were (100%) consistent with the results identified by ISO standard. Conclusions Vibriocould be accurately identified at the species level by using the ITS sequences. Significance and Impact of the Study The present study suggests that the ITS can be considered as a significant DNA marker for identification and classification ofVibriospecies, and it posed a new path to screen theVibrioin food sample.