Desorption electrospray ionization (DESI) is a powerful and versatile ambient ionization surface analysis technique. In this ionization, molecules are firstly desorbed and then ionized from the surface under ambient conditions. Mass spectrometry imaging (MSI) is used to elucidate chemical distributions of samples and to provide spatial context. An MSI workflow is clear. There are four steps for basic MSI: Sample selection and preparation, Desorption and ionization, Mass analysis and Image registration and data analysis. DESI is an ambient ionization technique used for the mass spectrometric imaging of samples. DESI-MSI helps to identify the fundamental biology, chemical structure, and surface metabolites, and it is applied to measure stress responses. DESI-MSI has successfully been used as a rapid method for the trace analysis of components in food. DESI-MSI research will promote the future study for the development of new techniques and will supply instrumentation for chemical mapping of foods.