Brettanomyces bruxellensis is the most reported spoilage yeast in aged wines mainly due to the production of phenolic off-flavors. In the present study, 64 B. bruxellensis strains isolated from Catalonian ageing wines were genetically and physiologically evaluated. The B. bruxellensis strains had high intraspecific diversity and were distributed genetically using the polymerase chain reaction of the intron splice site (ISS-PCR) into 8 clusters, mostly depending on their origin, and into 22 clones using microsatellite analysis. Wine-like conditions resulted in poor growth of several strains but, those growing, increased their tolerance to sulfur dioxide (SO2) with incubation time. However, tolerance to SO2 was not related to the genetic clusters as defined using ISS-PCR. Furthermore, some of the strains were resistant and grew in 60 mg/L of total SO2 (2.67 mg/L molecular SO2) in wine-like conditions. Additionally, the spoilage potential of the isolated strains was evaluated using precursors of 4-ethylphenol and 4-ethylguaiacol. All the strains were able to produce these compounds above their detection threshold even if the cells were losing culturability with incubation time. Thus, both the resistance to SO2 and the production of volatile phenols varied within the strains. This complexity must be taken into account to optimize both monitoring and corrective interventions, suggesting the importance of an early detection.