Red yeast rice fermented by Monascus contains secondary metabolites such as monacolin K and azaphilone pigments. To date, no information on the localization of these secondary metabolites in red yeast rice has been reported. In this study, the localization of monacolin K, monascin, and rubropunctamine, produced by Monascus pilosus, was revealed through the use of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Monacolin K was visualized as being distributed throughout the rice during the fermentation process. By contrast, monascin and rubropunctamine were found to be localized near the surface of the rice. In addition, high-resolution imaging revealed that monacolin K was localized in the ascocarp of Monascus. This is the first such study to reveal the localization of secondary metabolites in red yeast rice during fermentation using MALDI-MSI. These findings will enhance our understanding of metabolite production in fungi.