Fungicides must penetrate the internal tissues of plants to kill pathogenic fungi. Mass spectrometers have been used to confirm this penetration, but conventional mass spectrometric methods cannot distinguish the fungicides in different internal tissues owing to the extraction steps. However, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) can detect the penetration of fungicides into leaf sections through direct analysis of the sample surfaces. Therefore, the objective of this study was to establish a method for visualizing fungicide penetration in wheat leaf cross sections using MALDI-MSI. The penetration of azoxystrobin from the epidermal to the internal tissue of the leaves was observed. Moreover, azoxystrobin accumulates in the cells around the vascular bundle. This study suggests that MSI can be useful for the evaluation of fungicide penetration in plant leaves.