ABSTRACT: Ellagitannins (ETs) are hydrolysable tannins composed of a polyol core, primarily glucose, which is esterified with hexahydroxydiphenic acid (HHDP), and in some cases, gallic acid. ETs are the major phenolic compounds found in strawberries and may contribute to the health-related properties of strawberries, because of their strong antioxidative activity. However, their distribution in the strawberry fruit remains unclear. In this study, matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize ETs in ripe strawberry fruits. Five peaks, corresponding to the m/z values of ET [M-H]- ions detected in the MALDI-MS spectrum of strawberry extracts, were identified as strictinin, pedunculagin, casuarictin, davuriicin M1, and an unknown ET using MALDI-tandem MS (MS/MS). In addition, liquid chromatography-electrospray ionization-MS/MS of the extracts revealed the presence of pedunculagin isomers and the unknown ET. Ion images of these five ETs were reconstructed using MALDI-MSI. Strictinin was widely distributed in and around the achene seed coats, while the other ETs were dispersed in and around the seed coats, and at the bottom of the receptacle; pedunculagin was distributed in the epidermis and pith, whereas casuarictin, the unknown ET, and davuriicin M1 were distributed in the pith. Moreover, MALDI-MSI of a casuarictin standard indicated that in-source fragmentation weakly affected the ion images. The results suggest that the distribution of ETs depends on the presence or absence of their constituents, namely galloyl units, HHDP, and bis-HHDP. To the best of my knowledge, this is the first report on the visualization of ETs in plant tissues using MSI, MALDI-MSI may be a useful tool for analyzing the distribution of ETs in the strawberry fruit.