Covering: up to the end of September 2013 Microorganisms are a plentiful resource for natural products research. Traditionally, natural products discovery from microbial sources depends on the screening of target-mediated inhibition. The natural products identified through this strategy usually correlate to significant microbial phenotypes. However, the target-mediated transcriptions deduced from low concentrations of natural products sometimes do not generate an obvious phenotype. The better understanding of the true biological roles of those microbial natural products will permit the application of rational approaches to the more effective exploitation of their use. Imaging mass spectrometry (IMS) has been developed and applied in many fields for decades. However, the applications of IMS on microbial natural products research have just been recently reported. IMS is one of few tools capable of revealing both phenotype and relevant and irrelevant chemotypes of microorganisms. In this review, we summarize the latest applications of IMS technologies. The challenges and prospect of improvement and application of IMS to microbial natural products research are discussed as well.