Efficacy of potential treatments for myocardial infarction (MI) is commonly assessed by histological measurement of infarct size in rodent models. In experiments involving an acute MI setting, measurement of the infarcted area in tissue sections of the left ventricle is a standard approach to determine infarct size. This approach has also been used in the chronic infarct setting to measure infarct area several weeks post-MI. We tested the hypothesis that, because wall thinning is known to occur in the chronic setting, the area measurement approach would be less appropriate. We compared infarct measurements in tissue sections based on 1) infarct area, 2) epicardial and endocardial infarct arc lengths, and 3) midline infarct arc length. Infarct sizes from all three measurement approaches correlated significantly with left ventricular ejection fraction and wall motion abnormality. However, the infarct size values derived from the area measurement approach were significantly smaller than those from the other two measurement approaches, and the range of values obtained was compressed 0.4-fold. The midline method allowed detection of the expected size differences between infarcts of variable severity resulting from proximal vs. distal ligation of the coronary artery. Segmental infarct size was correlated with segmental wall motion abnormality. We conclude that both area- and length-based measurements can be used to determine relative infarct size over a wide range of severity, although the area-based measurements are substantially more compressed due to wall thinning, and that the estimation of infarct midlines is a simple, reliable approach to infarct size assessment.