Near-infrared spectroscopic imaging (NIRSI) is useful to assess cardiac tissue oxygenation in arrested and beating hearts, and it shows potential as an intraoperative gauge of the effectiveness of bypass grafting. The purpose of this study was to determine whether NIRSI can reliably differentiate among a range of cardiac oxygenation states, using ischemia and hypoxia models independently. An ischemia-reperfusion model was applied to isolated, beating, blood-perfused porcine hearts, in which the left anterior descending (LAD) artery was cannulated. LAD flow was decreased stepwise to approximately 50, 20, and 0% of normal flow and was completely restored between ischemic episodes. Upon completion of the ischemia-reperfusion protocol, the hearts were further subjected to periods of increasingly severe global hypoxia. Regional oxy- and deoxy-hemoglobin (myoglobin) levels were derived from spectroscopic images (650 to 1050 nm) acquired at each step. Oxygenation maps vividly highlighted the area at risk for all degrees of ischemia. Oxygenation values differed significantly for different LAD flow rates, regardless of whether intermediate reperfusion was applied, and oxygenation values during progressive hypoxia correlated well with blood oxygen saturation. These results suggest that NIRSI is well suited, not only to identify ischemic or hypoxic regions of cardiac tissue, but also to assess the severity of deoxygenation.