Mass spectrometry imaging (MSI) is an emerging technology for high-resolution plant biology. It has been utilized to study plant–pest interactions, but limited to the surface interfaces. Here we expand the technology to explore the chemical interactions occurring inside the plant tissues. Two sample preparation methods, imprinting and fracturing, were developed and applied, for the first time, to visualize internal metabolites of leaves in matrix-assisted laser desorption ionization (MALDI)-MSI. This is also the first time nanoparticle-based ionization was implemented to ionize diterpenoid phytochemicals that were difficult to analyze with traditional organic matrices. The interactions between rice–bacterium and soybean–aphid were investigated as two model systems to demonstrate the capability of high-resolution MSI based on MALDI. Localized molecular information on various plant- or pest-derived chemicals provided valuable insight for the molecular processes occurring during the plant–pest interactions. Specifically, salicylic acid and isoflavone based resistance was visualized in the soybean–aphid system and antibiotic diterpenoids in rice–bacterium interactions.