Instability of the inner mitochondrial membrane potential (ΔΨ_m) has been implicated in electrical dysfunction, including arrhythmogenesis during ischemia-reperfusion. Monitoring ΔΨ_m has led to conflicting results, where depolarization has been reported as sporadic and as a propagating wave. The present study was designed to resolve the aforementioned difference and determine the unknown relationship between ΔΨ_m and electrophysiology. We developed a novel imaging modality for simultaneous optical mapping of ΔΨ_m and transmembrane potential ( V_m). Optical mapping was performed using potentiometric dyes on preparations from 4 mouse hearts, 14 rabbit hearts, and 7 human hearts. Our data showed that during ischemia, ΔΨ_m depolarization is sporadic and changes asynchronously with electrophysiological changes. Spatially, ΔΨ_m depolarization was associated with action potential duration shortening but not conduction slowing. Analysis of focal activity indicated that ΔΨ_m is not different within the myocardium where the focus originates compared with normal ventricular tissue. Overall, our data suggest that during ischemia, mitochondria maintain their function at the expense of sarcolemmal electrophysiology, but ΔΨ_m depolarization does not have a direct association to ischemia-induced arrhythmias.