Terminal restriction fragment length polymorphism (TRFLP), used most often to describe bacterial communities, presents a high-throughput, low-cost solution for analyzing mixed yeast communities in wine and other fermentations. In this study, a TRFLP approach was developed for the identification and discrimination of yeasts and used to construct a TRFLP database comprising 121 strains of yeast (representing 24 genera and 72 species) associated with wine and food fermentations. This database exhibits sensitive discrimination among species and robust intraspecific conservation of TRFLP profiles, enabling reliable characterization of mixed yeast communities. The yeast ecology of sweet, botrytized wine fermentations from two separate vintages was analyzed using this database, demonstrating the utility of this method for fast-paced, qualitative detection and identification of differences in yeast community structures over time in a complex, diverse fermentation system.