BACKGROUND Anomalous coronary arteries originating from the opposite sinus of Valsalva (ACAOS) are associated with adverse cardiac events. Discrimination between ACAOS and coronary artery disease (CAD)-related perfusion defects may be difficult. The aim of the present study was to investigate the value of hybrid coronary computed tomography angiography (CCTA)/SPECT-MPI in patients with ACAOS and possible concomitant CAD. METHODS We retrospectively identified 46 patients (mean age 56 ± 12 years) with ACAOS revealed by CCTA who underwent additional SPECT-MPI. ACAOS with an interarterial course were classified as malignant, whereas all other variants were considered benign. CCTA/SPECT-MPI hybrid imaging findings (ischemia or scar) were analyzed according to the territory subtended by an anomalous vessel or a stenotic coronary artery. RESULTS Twenty-six (57%) patients presented with malignant ACAOS. Myocardial ischemia or scar was found only in patients who had concomitant obstructive CAD in the vessel matching the perfusion defect as evidenced by hybrid CCTA/SPECT imaging. CONCLUSION Hybrid CCTA/SPECT-MPI represents a valuable non-invasive tool to discriminate the impact of ACAOS from concomitant CAD on myocardial ischemia. Our results suggest that in a middle-aged population myocardial ischemia due to ACAOS per se may be exceedingly rare and is more likely attributable to concomitant CAD.