A large number of plant metabolites were discovered, but their biosynthetic and metabolic pathways are still largely unknown. However, the spatial distribution of metabolites and their changes in metabolic pathways can be supplemented by mass spectrometry imaging (MSI) techniques. For this purpose, the combination of desorption electrospray ionization (DESI)-MSI and non-targeted metabolomics was used to obtain the spatial distribution information of metabolites in the leaves of Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus). The sample pretreatment method was optimized to have higher detection sensitivity in DESI. The changes of metabolites in C. paliurus were analyzed in depth with the integration of the spatial distribution information of metabolites. The main pathways for biosynthesis of flavonoid precursor and the effect of changes in compound structure on the spatial distribution were found. Spatial metabolomics can provide more metabolite information and a platform for the in-depth understanding of the biosynthesis and metabolism in plants.