Two lager yeast strains were used to study the cell morphology, cell viability and vitality, reactive oxygen species (ROS) and DNA damage so as to analyze the changes of physiological properties of lager yeasts during serial re-pitching and autolysis. The results showed that, lager yeast cells with full and round surface and plump shape became wrinkled during serial re-pitching, and even withered out because of the severe hydrolysis of the inner structure of cells. At the same time, the thickness of cell wall gradually became thinner during serial re-pitching, which affected the morphology of the cells. Lager yeast with better anti-autolytic ability showed higher vitality, better stress resistance, lower ROS accumulation, and less DNA damage. Therefore, it is of great significance to understand the physiological changes and autolysis performances of lager yeast during serial re-pitching, which will helpful for understanding the self-defense mechanism of yeast cells during serial re-pitching. In the meantime, the findings from this study has important guiding significance for breeding of brewer's yeasts.