The control of wine quality is related also to the knowledge of composition and dynamics of yeast-flora occurring throughout the vinification process. The restriction analysis of NTS and ITS regions was used to differentiate in mixed fermentation non-Saccharomyces species frequently encountered in the early fermentation phase, such as Hanseniaspora uvarum, Candida stellata and Metschnikowia pulcherrima. PCR-RFLP analysis of ITS region allowed to identify all the species inoculated in the grape must. The novelty of the protocol used is the application of PCR-RFLP analysis on yeast DNA isolated directly from grape must fermentation, without previous yeast colony isolation. Key words: Non-Saccharomyces yeasts, NTS region, yeast mixed cultures.