Tostado wine is a prestigious sweet wine produced in Galicia (NW Spain) from air-dried grapes of traditional grape varieties grown in this region. The peculiar characteristics of the must, mainly its high sugar concentration, make its fermentation particularly difficult. The aim of this study was to characterize the dynamics of the yeast populations involved in Tostado fermentation. White and red Tostado musts, supplied by local winegrowers, were allowed to ferment spontaneously. Yeasts were isolated at different stages of the process using conventional methodology. The isolated yeasts were then identified to the species level by PCR amplification of the 5.8S rRNA gene and the two internal spacers ITS1 and ITS2. Saccharomyces cerevisiae isolates were characterized at the strain level by mtDNA-RFLP. The results showed the diversity of the yeast population during fermentations. Non-Saccharomyces yeasts, including Hanseniaspora uvarum, Metschnikowia spp., Starmerella bacillaris, and Candida spp., predominated in the early stages of fermentation; as fermentation progressed, codominance between different S. cerevisiae strains was observed. However, one trial fermented only with non-Saccharomyces species, mainly Lachancea thermotolerans and Starmerella bacillaris. The oenological properties of the isolated non-Saccharomyces species and S. cerevisiae strains will be analyzed to select suitable starter cultures for Tostado production.