Purpose : To compare the accuracy of digital subtraction images acquired by two different methods different in positioning four reference points for geometrical standardization. Materials and Methods : A total of 36 digital radiographic images of 6 volunteers were taken at the areas of the incisor, premolar, and molar of both the maxilla and mandible using the Digora system. Each image was moved 4 mm vertically and horizontally. Four oral and maxillofacial radiologists performed digital subtraction radiography between the paired images before and after movement using Emago (Oral Diagnostic Systems, Amsterdam, The Netherlands) and Sunny (Biomedisys Co., Seoul, Korea). The standard deviation of the internal gray value in Region of Interest (ROI) was statistically analyzed between the two programs using the paired t-test. Results : The standard deviation of pixel gray values from the digital subtraction images using the Sunny program were lower than that of the Emago program (p<0.05). All observers showed significant differences between each other when the Sunny program was used (p<0.05), but one observer showed a significantly higher score than other observers when they used Emago (p<0.05). The standard deviations of premolar area from both Sunny and Emago programs were significantly higher than those of anterior and molar regions (p<0.05). Conclusion: The subtracted images using the Sunny program were more accurate and sensitive than those taken using the Emago program.