Aims: Although numerous physiological and molecular methods have been proposed for yeast taxonomy, the unambiguous separation of Saccharomyces sensu stricto species in natural samples is still an incompletely resolved issue. In this study the power of various methods was compared in the identification of strains isolated from fermenting botrytized grape musts. Methods and Results: Conventional taxonomic and physiological tests and molecular methods developed for rapid identification were used. Conclusions: None of the methods tested was sufficiently powerful. However, the combination of electrophoretic karyotyping and the PCR-RFLP of MET2 with growth tests at 10 and 37degreesC provided results sufficient for species identification of Saccharomyces wine strains which were not interspecific hybrids or recombinants. Significance and Impact of the Study: The proposed combination of molecular and physiological methods allows specific taxonomic identification and separation of Saccharomyces wine strains without extensive genetic and molecular analysis. The proposed combined approach can also identify hybrids and recombinants.