In recent years, Brettanomyces/Dekkera bruxellensis has caused increasingly severe quality problems in the wine industry. A typing method at the strain level is needed for a better knowledge of the dispersion and the dynamics of these yeasts from grape to wine. Three molecular tools, namely random-amplified polymorphic DNA, PCR fingerprinting with microsatellite oligonucleotide primers and SAU-PCR, were explored for their relevance to typing strains of Brettanomyces bruxellensis. The results indicated that discrimination of each individual strain was not possible with a single PCR typing technique. We described a typing method for B. bruxellensis based on restriction enzyme analysis and pulse field gel electrophoresis (REA-PFGE). Results showed that electrophoretic profiles were reproducible and specific for each strain under study. Consequently, REA-PFGE should be considered for the discrimination of B. bruxellensis strains. This technique allowed a fine discrimination of B. bruxellensis, as strains were identified by a particular profile. This study constitutes a prerequisite for accurate and appropriate investigations on the diversity of strains throughout the winemaking and ageing process. Such studies will probably give clearer and more up-to-date information on the origin of the presence of Brettanomyces in wine after vinification when they are latent spoilage agents.