We sought to investigate the release pattern of different cardiac biomarkers (high-sensitivity C-reactive protein, cardiac troponin I, heart-type fatty acid-binding protein, creatine kinase-MB, and myoglobin) and to establish the diagnostic discrimination limits of each marker protein to evaluate perioperative myocardial injury in patients undergoing coronary artery bypass grafting with or without cardiopulmonary bypass. Fifty patients were randomly assigned to on-pump or off-pump coronary artery bypass grafting. All cardiac biomarkers were measured in serial venous blood samples drawn before heparinization in both groups and after aortic unclamping at 1, 2, 4, 8, 24, 48, and 72 hours in the on-pump group. In the off-pump group samples were taken after the last distal anastomosis and at same time intervals as in the on-pump group. The total amount of heart-type fatty acid-binding protein, cardiac troponin I, and high-sensitivity C-reactive protein released was significantly higher in the on-pump group than in the off-pump group. Receiver operating characteristic curve analysis of cardiac biomarkers indicated cardiac troponin I and heart-type fatty acid-binding protein as the superior diagnostic discriminators of myocardial injury, with an optimal cutoff value of greater than 0.92 ng/mL (area under the curve, 0.95 [95% CI, 0.88-1.00]; sensitivity, 92%; specificity, 92%; likelihood ratio [+], 11.50) and greater than 6.8 ng/mL (area under the curve, 0.94 [95% CI, 0.88-1.00]; sensitivity, 88%; specificity, 88%; likelihood ratio [+], 7.33), respectively. Logistic regression analysis revealed that patients with increased cardiac troponin I levels of greater than 0.92 ng/mL and heart-type fatty acid-binding protein levels of greater than 6.8 ng/mL were at 132.25 (95% confidence interval, 17.14-1020.49) times and 53.77 (95% confidence interval, 9.76-296.12) times higher risk of myocardial injury after on-pump coronary artery bypass grafting. Off-pump coronary artery bypass grafting provides better myocardial protection than on-pump coronary artery bypass grafting. Cardiac troponin I and heart-type fatty acid-binding protein, but not high-sensitivity C-reactive protein, served as superior diagnostic discriminators of perioperative myocardial damage after on-pump coronary artery bypass grafting.