Mass spectrometry imaging is an imaging technology that allows the localization and identification of molecules on (biological) sample surfaces. Obtaining the localization of a compound in tissue is of great value in biological research. Yet, the identification of compounds remains a challenge. Mass spectrometry alone, even with high-mass resolution, cannot always distinguish between the subtle structural differences of isomeric compounds. This review discusses recent advances in mass spectrometry imaging of lipids, steroid hormones, amino acids and proteins that allow imaging with isomeric resolution. These improvements in detailed identification can give new insights into the local biological activity of isomers.