Yeast is the major microorganism used to produce ethanol by fermentation. Because fermentation environments are stressful, the fermentation ability of yeast depends greatly on the yeast’s stress tolerance. Stress-tolerant yeasts can produce ethanol in a harsh environment. During the fermentation process of ethanol production, yeasts can be exposed to various stresses, including high temperature, hyperosmolarity, and/or inhibitory substances. The isolation of yeasts tolerant to these stresses and the clarification of their tolerance mechanisms are important for the development of stress-tolerant yeasts for efficient ethanol production. This review focuses on the stress tolerance of yeasts and the use of self-cloning-based gene modification to enhance yeast stress tolerance. We first conducted a search for stress-tolerant yeasts among our stock strains. We also identified stress tolerance-related genes using a Saccharomyces cerevisiae gene deletion strain collection. We then investigated the effects of stress tolerance-related gene overexpression on yeast growth and ethanol production.