Aims To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. Methods and Results Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V.similar to vulnificus, V.similar to cholerae and V.similar to alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22 degrees C (446 strains) and 37 degrees C (515 strains) in September 2009. The predominance of V.similar to alginolyticus (82.6%) among the Vibrio spp. strains isolated at 37 degrees C was shown. At 22 degrees C, only 1.6% of the strains were identified by PCR and they were V.similar to alginolyticus. Conclusions Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37 degrees C. For environmental strains isolated at 22 degrees C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. Significance and Impact of the Study The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.