The aim of this study was the development of miso with high quality and value by mutation breeding of koji molds with superior characteristics. We selected mutants generated by MNNG treatment of koji molds, and obtained two kinds of applicable strains for miso production. One is a high vitamin B2-producing strain with high neutral protease activity (R2 strain), which is useful for improving the color and taste of miso. The other is a high lipase-producing strain (NT12 strain), which increases miso functionality. We compared each mutant to BF-1, the commercial koji mold used as the control strain, during both laboratory-scale and pilot-scale miso fermentation. The koji manufactured from the R2 mutant showed high protease activity and contained 2∼3 times more vitamin B2 that manufactured from BF-1. In addition, the miso from the R2 strain showed higher x and y color values than that from BF-1, although the Y value (%) was somewhat lower. It also contained high contents of both formol and water-soluble nitrogen, and its color brightness and umami taste were evaluated well. On the other hand, the koji from the NT12 mutant showed lipase activity about 3 times higher than that from BF-1. The resulting miso using the NT12 strain was high in fatty acid ethyl esters, and showed higher inhibitory effects on the growth of tumor cells in vitro compared to BF-1.