Spontaneous fermentations are completed by a complex succession of non-Saccharomyces yeast species and S. cerevisiae strains that influence the final wine aroma and flavor profile. Regional yeast composition may help shape the wine characteristics typical in a region, a concept known as microbial terroir. To establish the yeast composition of Riesling grapes in the Finger Lakes American Viticultural Area (AVA), an ecological survey of vineyard and winery microbiome, with a particular focus on S. cerevisiae strains, was performed. Grapes, winery equipment, and spontaneous fermentations were sampled during the 2015 and 2016 vintages at three Finger Lakes AVA vineyards and the associated wineries. Yeast was isolated using culture-dependent methods, identified using the 5.8S internal transcribed spacer (ITS) rRNA region, and S. cerevisiae strains were characterized using a six-locus multiplex variable number of tandem repeat (VNTR) analysis. Species from the Aureobasidium, Candida, Hannaella, Hanseniaspora, Metschnikowia, Meyerozyma, Pichia, Rhodotorula, Saccharomyces, Torulaspora, Trigonopsis, Wickerhamomyces, Zygoascus, and Zyosaccharomyces genera were identified. Numerous unrelated native S. cerevisiae strains, and a small number of commercial strains, were commonly observed in fermentations. When commercial strains were present, they did not always become established or dominant in the fermentation, and never completely displaced the native strains. Several native S. cerevisiae strains are likely part of the regional microbiome and should be investigated further. The S. cerevisiae strains observed in fermentations appear to be influenced by regional and resident winery microbiome, and by vintage specific factors.