The purpose of this study was to investigate the ability of digital subtraction radiography (DSR) to reveal anatomic features of C-shaped canal systems in mandibular second molars with the aid of an intraradicular contrast medium. Thirty mandibular second molars with fused roots were collected and stored in 10% formalin solution. After being scanned with microcomputed tomography (μCT) 20 and reconstructed, the root canals were mechanically and chemically cleaned of their pulp tissue. Buccal-lingual digital radiographic (radiovisiography [RVG]) images were taken with mandibular bone plates for each tooth. After the introduction of contrast medium into canals, RVG images were taken again in the same way as before. The digital images taken before and after contrast medium introduction were overlapped, and the image subtraction was performed by using the 3D Doctor software (Able Software Corp, Lexington, MA). All the subtracted canal images (DSR) and reconstructed canal images based on μCT scanning were classified into three categories (ie, type I, II, and III). The classifications based on reconstructed canal images were used as the standard to evaluate the sensitivity (Se) and specificity (Sp) of DSR classification. The results were subjected to the Wilcoxon statistical test, which showed there was no significant difference between the two classifications (p > 0.05). The DSR classification had a very high Se and Sp in all three categories. It is feasible to use the DSR technique clinically to identify anatomic features of the C-shaped canal system in mandibular second molars.