Aims: To identify and describe the indigenous yeast population involved in traditional balsamic vinegar (TBV) fermentation. Methods and Results: Using the restriction analysis of the ribosomal region 5(.)8S (5.8S rRNA) and the internal transcribed spacers 1 and 2 (5(.)8S-ITS region) we were able to group 133 strains isolated from 17 cooked grape must samples into 10 different yeast species, included into 4 genera. Moreover, we sequenced the D1/D2 domains of the 26S rRNA and confirmed the reliability of each identification at species level. Most strains belonged to the genus Zygosaccharomyces. In particular, Zygosaccharomyces bailii was found in 41% of the samples, followed by Saccharomyces cerevisiae, Zygosaccharomyces pseudorouxii and Candida stellata. Strains belonging respectively to Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, Hanseniaspora valbyensis, Hanseniaspora osmophila and Candida lactis-condensi species were also detected. Despite the great number of species recovered, the mtDNA restriction profiles showed low variability at strain level. Saccharomyces cerevisiae isolates with an higher degree of intraspecific variance were considered an exception. Conclusions: Many different indigenous yeast species were recovered and TBV yeasts population seems to be far more complex than what was reported in previous literature. Significance and Impact of the Study: This study has allowed us to gain a better understanding of the indigenous yeast species of TBV cooked must.