<p>Nitric oxide, hydrogen sulfide and carbon monoxide are bona fide gasotransmitters with multifaceted, concentration-dependent actions that are often context specific. In healthy tissues, these gasotransmitters play a role in maintaining homeostasis but, under pathological conditions, the gaseous molecules can have either protective or damaging effects depending on their location and concentration. Investigation of the paradoxical effects of gasotransmitters in vivo is limited by their picomolar-to-micromolar abundance as well as their rapid diffusion and half-lives of seconds to minutes. However, technological advances in fluorescence, photoacoustics, chemiluminescence, afterglow, bioluminescence, magnetic resonance and radionuclide imaging permit real-time visualization of gasotransmitter dynamics in intact organisms. In this Review, we analyse these imaging modalities with a focus on probe chemistry, sensitivity, spatiotemporal resolution and quantitative readouts. By addressing remaining challenges, such as insufficient selectivity in complex redox environments, the requirement for second-level temporal resolution and the scarcity of clinically viable probes, we can evaluate the clinical potential of gasotransmitter imaging. Spatiotemporal maps of nitric oxide, hydrogen sulfide and carbon monoxide, developed through the integration of molecular imaging and systems biology, hold promise for illuminating disease mechanisms, informing therapeutic development and advancing the progression toward precision medicine.</p>