In sake brewing, the quality of rice koji is evaluated by experienced sake brewers based on visual inspection of the haze, which is defined by the extent of fungal hyphae spread on/into the rice grains. There is an increasing interest in understanding the factors that affect the quality of rice koji, which is dependent on its making process. Several studies have focused on the degree of mycelial penetration (haze-komi) and enzyme production during rice koji production. However, there are limited analytical methods available to monitor hyphal growth on a solid surface. Here we used a beta-glucuronidase (GUS)-expressing strain of Aspergillus oryzae to visualize and map the fungal growth on rice koji grains. Observation of indigo color revealed that A. oryzae hyphae penetrated the steamed rice grain in the early stage (24 h) of rice koji-making before spreading on the surface during the later stages. Additionally, hyphae penetrated along the endosperm cells and penetrated the cells to form the sou-haze. Furthermore, mass spectrometry imaging (MSI) of glucose demonstrated that the area of mycelial penetration is directly correlated with the spread of glucose during fermentation. This is the first report on utilizing new tools such as GUS-body-mapping of A. oryzae and MSI to monitor fungal growth during rice koji making. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.