In this study, we identified Fis1p as the factor responsible for mitochondrial fragmentation during sake brewing and showed that inhibiting mitochondrial fragmentation causes higher malate production in both laboratory and industrial strains. These findings indicate that mitochondrial fragmentation plays an important role in the metabolism of constituents during brewing, and at the same time, provides a potent strategy for the isolation of a high-malate-producing sake yeast. The effect of morphological alteration of yeast mitochondria during alcohol fermentation on fermentation characteristics had not been resolved until the present study.