This study regards yeasts isolated from refrigerated musts with patent ongoing fermentation. The isolates belonged to the genus Saccharomyces sensu stricto with an optimal growth temperature less than 30 °C. Karyotype analysis highlighted the constant presence of two well-defined trait bands in the low molecular weight between 225 and 375 kb that distinguished them unequivocally from normal Saccharomyces cerevisiae strains. The isolated strains individually reinoculated in sterile must at 25 °C, produce complete fermentation. Analysis of minor fermentation compounds revealed other differences between these strains and Saccharomyces cerevisiae strains. These strains are responsible for the spoilage of fermenting refrigerated must. Phenotype traits, combined with karyotype, suggest that they should not be assigned to Saccharomyces cerevisiae but more reasonably to Saccharomyces bayanus uvarum type.