Ambient mass spectrometry (AMS)-based techniques are performed under ambient conditions in which the ionization and desorption occur in the open environment allowing the direct analysis of molecules with minimal or no sample preparation. A selected group of AMS techniques demonstrate imaging capabilities that can provide information about the localization of molecules on complex sample surfaces such as biological tissues. 2D, 3D, and multimodal imaging have unlocked an array of applications to systematically address complex problems in many areas of research such as drug monitoring, natural products, forensics, and cancer diagnostics. In the present review, we summarize recent advances in the field with respect to the implementation of new ambient ionization techniques and current applications in the last 5 years. In more detail, we mainly focus on imaging applications in topics related to animal whole bodies and tissues, single cells, cancer diagnostics and biomarkers, microbial cultures and co-cultures, plant and natural product metabolomics, and forensic applications. Finally, we discuss new areas of research, future perspectives, and the overall direction that the field may take in the years to come.