The composition of wine yeast populations, present during spontaneous fermentation of Chardonnay, Pinot Gris and Riesling from the Lake Erie Region was studied. A combination of biochemical and molecular techniques was used to identify non-Saccharomyces and Saccharomyces yeast isolates. The biochemical techniques included analysis of yeast isolates by sugar fermentation and carbon and nitrogen assimilation. Molecular techniques involved ribotyping of a highly variable segment in the 26S rRNA gene using DNA sequence analysis and restriction fragment length polymorphism of amplified DNA. The results show that of the non-Saccharomyces yeasts, several related species of Hanseniaspora, were the most abundant yeasts present during early stages of fermentation. Later in fermentation S. cerevisiae dominated, and based on biochemical analyses consisted of a heterogeneous group of genotypes. There were no major differences in yeast populations among the three types of juice analyzed.