The aim of this study was (i) to investigate changes occurring in the yeast population profile during spontaneous fermentation of grape juice; (ii) to assess the proliferation of commercial yeast starter culture strains in vineyards; and (iii) to identify indigenous wine strains for future development of starter strains that better reflect the yeast biodiversity of China's grape-growing regions. To achieve this, yeasts were isolated at four different stages during fermentation of both hand-pressed and winery-sourced must samples of Vitis vinifera L. cv. Roussanne and Merlot. A total of 1600 yeast colonies were isolated and then grouped according to macroscopic and microscopic characteristics. A selection of 291 colonies from the different groups was subjected to species identification using the internal transcribed spacer regions of the 5.8S rRNA gene (ITS1-5.8S-ITS2 region) and the inter-delta (δ) sequence of the 26S rRNA D1/D2 region. In addition, 104 Saccharomyces cerevisiae colonies were subjected to strain identification. Twelve species belonging to nine different genera were found amongst the isolates. During the early stages of fermentation, it was found that Hanseniaspora uvarum and Candida stellata numerically dominated the four to six yeast species present, including a region-specific yeast, Sporobolomyces beijingensis. Two S. cerevisiae strains were isolated from the final stage of fermentation. These two indigenous strains, which were found to be different from the nine commercial yeast strains previously used as starter cultures in this particular Beijing-based winery, might possess potentially important region-specific oenological characteristics. This study provides the first essential step towards the preservation and exploitation of the hidden oenological potential of the untapped wealth of yeast biodiversity in China's wine-producing regions.