Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) is a technique that records mass spectra as a function of position across a biological tissue sample, yielding images of chemical distribution. Until now, MALDI-MSI has typically been performed on thinly sliced tissue sections that are coated with a UV-absorbing matrix. We have developed protocols to apply MALDI-MSI to chemically interrogate intact Caenorhabditis elegans, a nematode approximately 1-mm in length. C. elegans is a model organism with numerous available genetic mutants, three of which were used in this study to validate the MALDI-MSI results using principal component analysis (PCA). In comparison to traditional chemical biology analyses of nematodes that require large-scale cultures, MALDIMSI has the selectivity and sensitivity to record chemically relevant data from analysis of a single worm. This study demonstrates the feasibility of MALDI-MSI as an important new tool to study the chemistry of individual nematodes as well as the potential to conduct chemical biology and metabolomics studies of parasitic species that are impossible to culture outside of the host.