EBT2 film is widely used for quality assurance in radiation therapy. The purpose of this study was to investigate the homogeneity of EBT2 film among various lots, and the dose dependence of heterogeneity. EBT2 film was positioned in the center of a flatbed scanner and scanned in transmission mode at 75 dpi. Homogeneity was investigated by evaluating gray value and net optical density (netOD) with the red color channel. The dose dependence of heterogeneity in a single sheet from five lots was investigated at 0.5, 2, and 3 Gy. Maximum coefficient of variation as evaluated by netOD in a single film was 3.0% in one lot, but no higher than 0.5% in other lots. Dose dependence of heterogeneity was observed on evaluation by gray value but not on evaluation by netOD. These results suggest that EBT2 should be examined in each lot number before clinical use, and that the dose calibration curve should be constructed using netOD.