Photoacoustic imaging (PAI) is an evolving real-time imaging modality that combines the higher contrast of optical imaging with the higher spatial resolution of ultrasound imaging. We utilized dual-wavelength PAI for the diagnosis and monitoring of myocardial ischemia by assessing variations in blood oxygen saturation estimated in a murine model. The use of high-frequency ultrasound in conjunction with PAI enabled imaging of anatomic and functional changes associated with ischemia. Myocardial ischemia was established in eight mice by ligating the left anterior descending artery (LAD). Longitudinal results reveal that PAI is sensitive to acute myocardial ischemia, with a rapid decline in blood oxygen saturation (p ˂ 0.001) observed after LAD ligation (30 min: 33.05 ± 6.80%, 80 min: 36.59 ± 5.22%, 120 min: 36.70 ± 9.46%, 24 h: 40.55 ± 13.04%) compared with baseline (87.83 ± 5.73%). Variation in blood oxygen saturation was found to be linearly correlated with ejection fraction (%), fractional shortening (%) and stroke volume (µL), with Pearson's correlation coefficient values of 0.66, 0.67 and 0.77, respectively (p ˂ 0.001). Our results indicate that PAI has the potential for real-time diagnosis and monitoring of acute myocardial ischemia.