Vibrio vulnificus is a Gram-negative bacterial pathogen responsible for the vast majority of bacterially mediated fatalities from the consumption of raw or undercooked seafood in the USA. Vibrio vulnificus-associated septicaemia can occur rapidly (< 24 h); however, methods for the isolation and confirmation of V. vulnificus from seafood samples typically require several days. A real-time PCR assay was developed for V. vulnificus biotype 1 that provides a rapid means of identifying a gene fragment (vcgC) previously indicated as a strong predictor of potential virulence. PCR probe specificity was confirmed by amplification of 17 clinical V. vulnificus strains and by the lack of amplification with seven non-pathogenic V. vulnificus isolates and a wide range of closely related bacteria. Oyster and seawater samples were amended with a range of environmentally realistic concentrations of C-genotype V. vulnificus cells, which were quantitatively and unambiguously identified according to biotype. Of some significance, we utilized a sample processing and nucleic acid extraction procedure that allowed identification of pathogenic strains of V. vulnificus from oyster matrices without prior enrichment or culturing of strains. This outlined approach allowed the detection of as little as 50 cfu of V. vulnificus in less than 5 h, which compares favourably with culture-based approaches. The results indicate the applicability of this approach for monitoring purposes or as a potential diagnostic tool in clinical settings.