Question Rising sea water temperature due to global warming enhances the conditions for Vibrio spp. to grow and disperse even in temperate waters of the North and Baltic Sea. Because of the increased incidence of Vibrio infections in the last years, a rapid and accurate method is required to analyze and identify complex Vibrio spp. populations, specifically potential pathogenic Vibrio species, in environmental samples. A PCR-DHPLC (Denaturing High Performance Liquid Chromatography) has been developed based on the rpoB gene of the genus Vibrio, which is a promising method to not only identify but also separate Vibrio spp. in mixed samples due to the different running behavior of amplified PCR products. Methods To facilitate the identification of potential human-pathogenic species we designed Vibrio specific primers based on rpoB sequences of Vibrio spp. strains isolated at Helgoland Roads (North Sea). These primers were combined to amplify fragments of ~500 bp of this rpoB gene. Using the PCR products of four different Vibrio species, we systematically improved the DHPLC conditions, including column temperature and acetonitrile gradient. Finally, we compared the PCR-fragment separation with and without a 40-bp clamp attached to the amplification primers. Results and Conclusions We developed five primer-sets for different regions of the targeted rpoB gene and verified the primer-sets by successfully amplification of 20-23 different Vibrio species (in total 31). We could show that for optimal separation of the amplified fragments by DHPLC the column temperature plays a crucial role and needs to be adapted for each PCR-fragment individually, either with or without GC-clamp. The attached GC-clamp was essential for partial denaturing of all fragments at the same temperature, but generated ambiguous retention peaks. We will illustrate the development of this method in detail.