The yeast Brettanomyces bruxellensis , generally considered the main oenological spoilage microbe, is able to survive during the winemaking process and it confers off-odors to wine, in reason of its ability to produce considerable amounts of volatile phenols. Forty-eight isolates of B . bruxellensis , obtained from several wines collected in Apulia (Southern Italy), were genetically characterized using an integrated approach, including a strain biodiversity analysis by Sau -PCR. Furthermore, the production of volatile phenols was assessed in wine and in synthetic medium, confirming the oenological spoilage potential of the analysed strains. Our findings indicate a remarkable genetic variability of the B. bruxellensis identified strains and corroborate the evidence of a high level of genotypic and phenotypic polymorphism within B. bruxellensis species. Moreover, the observation reported suggest that strains from wines produced in the same geographical areas often clustered differently, indicating a complex intraspecific biodiversity in the regional wine environments. Diversity in volatile phenol production reflects intraspecific biodiversity highlighted by Sau -PCR. Strains diversity linked to differences in ‘spoilage potential’ increase the industrial relevance of this study, allowing the design of new strategies for B. bruxellensis control in wines.