The Gram-negative bacterium Vibrio vulnificus is a common inhabitant of estuarine environments. Globally, V. vulnificus is a significant foodborne pathogen capable of causing necrotizing wound infections and primary septicemia, and is a leading cause of seafood-related mortality. Unfortunately, molecular methods for the detection and enumeration of pathogenic V. vulnificus are hampered by the genetically diverse nature of this pathogen, the range of different biotypes capable of infecting humans and aquatic animals, and the fact that V. vulnificus contains pathogenic as well as non-pathogenic variants. Here we report an alternative approach utilizing the development of a real-time PCR assay for the detection of pathogenic V. vulnificus strains based on a polymorphism in pilF, a gene previously indicated to be associated with human pathogenicity. Compared to human serum reactivity, the real-time PCR assay successfully detected pathogenic strains in 46 out of 47 analysed V. vulnificus isolates (97.9%). The method is also rapid, sensitive, and more importantly can be reliably utilised on biotype 2 and 3 strains, unlike other current methods for V. vulnificus virulence differentiation.