Background and Aim: Conventional oral radiography poses unwanted limitations on proper detection of class III caries. The purpose of this study was to evaluate the accuracy of Digital Subtraction Radiography( DSC) in the diagnosis of class III caries with various depths.Materials and Methods: This diagnostic study assessed the depth of class III caries on 46 extracted intact permanent anterior human teeth. Direct digital radiography of samples were initially obtained under standard conditions, prior to preparation of manual artificial caries (group A). In each tooth, one proximal surface was randomly selected for creation of an artificial cavity using a 008 round diamond bur (RDB). At this stage, secondary radiographic images were obtained from the samples and they were categorized into (group B). The depth of the previously created caries was then increased by two fold using a 009 RDB (group C) and 010 RDB (group D) followed by secondary radiographic images obtained from each group. The presence or absence of caries was assessed by two examiners after obtaining subtraction images. Data analysis was done by SPSS 11.5 using diagnostic tests.Results: Positive predictive value, negative predictive value, sensitivity, specificity and accuracy of group B were 90%, 84%, 83%, 91% and 87%, respectively and that of group C were 96%, 94%, 93%, 96 and 95%, respectively. For group D all the indices were 100% (P<0.001).Conclusion: Digital Subtraction Radiography (DSR) can be used as a specialized and precise method in the diagnosis of class III caries.