Three main types of root canal sealer are currently commonly used in pulp treatment: zinc oxide eugenol-based, calcium hydroxide-based, and epoxy resin-based sealers. In the present study, the genotoxicity of sealer on oral carcinoma cells was evaluated by single-cell gel electrophoresis assay (comet assay). The whole length of the comet and the diameter of the head were measured using an image analysis system. The results were analyzed by one-way analysis of variance to compare the various means. The zinc oxide eugenol-based sealers (Canals, Canals-N, and Tubilseal) did not always cause a dose-dependent increase in genotoxicity. The resin-based sealers (Topseal, AH 26, and AH Plus) caused a dose-dependent increase in genotoxicity, but no such effect was seen with the calcium hydroxide-based sealer (Sealapex). The highest level of DNA damage was induced by the resin-based sealers.