The dynamics of the wine yeast strains presented in five spontaneous Malvasia wine fermentations have been studied. Samples were analysed for their microbiological characteristics and chemical substances. All 937 isolates were characterized using electrophoretic karyotyping and tested for their killer activity. The non- Saccharomyces population was identified using a combination of PCR-RFLP analysis of the rDNA spacer region and physiological testing. The total yeast population level in the must after sedimentation was 105cfu ml−1and included the following genera:Candida, Metschnikowia, Hanseniaspora, Rhodotorula, Issatchenkia and Debaryomyces. However, Saccharomyces sp. was not detected in fresh must samples plated on YEPD medium. Based on the chromosome length polymorphism among 649 isolates from the subsequent phases of fermentation, 46 different electrophoretic patterns of Saccharomyces cerevisiae were distinguished. The most abundant karyotypes were L1, L4, L12, P6. A sequential substitution of S. cerevisiae strains occurred during the different phases of fermentation. At the slow fermentation rate, karyotype L4was most abundant in almost all fermenters. At the beginning of the tumultuous fermentation phase, the most frequent karyotype became L1followed by karyotype L4. Finally, during the fermentation process, pattern L4was clearly replaced by karyotype L1followed by pattern L12. Despite the same fermentation source (grape must), differences among five spontaneous fermentations were observed. The population dynamics of S. cerevisiae yeasts, especially the dynamics of the major S. cerevisiae strains (L1, L4, and L12) were quite similar in all five fermenters in opposite to the minor strains of S. cerevisiae.