BACKGROUND: Although magnetocardiography (MCG) has been proposed as a non-invasive technique with high accuracy for functional diagnosis of myocardial injury, the validation of MCG against fractional flow reserve FFR in diagnosing coronary artery disease (CAD) has not yet been established. The goal of the study was to determine the diagnostic accuracy of MCG versus invasively determined FFR in patients with suspected or known CAD. METHODS: Forty seven patients with suspected CAD (35 men; mean age 69 years) who underwent coronary angiography and FFR measurement were enrolled. FFR ≤0.8 was considered to indicate significant myocardial ischemia. The change of ST-segment fluctuation score from rest to stress was calculated from the MCG. In addition, two blinded cardiologists assessed MCG images that were visualized by post-processing method, bull's-eye mapping. RESULTS: The best cut-off value of the percent change of ST-segment fluctuation score was −39.0% with sensitivity of 86.7% and specificity of 73.9%. Sensitivity, specificity, diagnostic accuracy, and the area under the receiver-operator characteristics curve of bull's-eye mapping for the detection of significant CAD were 90.5%, 92.3%, 91.5%, and 0.914 on a patient basis and 90.0%, 93.8%, 92.3%, and 0.919 by coronary territory, respectively. CONCLUSIONS: MCG accurately detects functionally significant CAD as defined by using FFR, provides an assessment of ischemic status in agreement with the change of ST-segment fluctuation score, and accurately localizes the ischemic territory in bull's eye mapping. Therefore, MCG may provide an incremental value for prediction of myocardial ischemia non-invasively and safely in clinical practice with fast examination time.