The accumulation of organic pollutants in vegetables is a major global food safety issue. The concentrations of pollutants in vegetables usually differ across different tissues because of different transport and accumulation pathways. However, owing to the limitations of conventional methods, in situ localization of typical organic pollutants such as phthalate esters (PAEs) in plant tissues has not yet been studied. Here, we developed a quick and efficient method for in situ detection and imaging of the spatial distribution of PAEs in a typical root vegetable, carrot, using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS). The use of a 2,5-dihydroxybenzoic acid matrix with a spray−sublimation coating method led to the successful identification of PAEs ion signals. The IMS results showed that a typical PAE−di-(2-ethylhexyl)phthalate (DEHP) was broadly distributed in the cortex, phloem, and metaxylem, but was barely detectable in the cambium and protoxylem. Interestingly, MALDI-IMS data also revealed for the first time the spatial distribution of sugars and β-carotene in carrots. In summary, the developed method offers a new and practical methodology for the in situ analysis of PAEs and plant metabolites in plant tissues. As a result, it could provide a more intuitive understanding of the movement and transformation of organic pollutants in soil−plant systems.