Mass spectrometry imaging (MSI) combined with matrix-assisted laser desorption/ionization (MALDI) is an efficient technology applied in plant metabolomics research. This technique allows for visualization of spatial distribution of metabolites such as: lipids, proteins, peptides and DNA sequences, by determining the x, y coordinates of the compounds exactly in plant tissue. Simplicity of the tissue preparation without the need of prior exact knowledge about the analytes is a great advantage of this method. In this review, we provide an overview of experimental workflow including sample preparation, data acquisition and analysis, methodology, and some recent applications of MALDI MS imaging in plant metabolomics research.