In vivo comet assays are often used to evaluate genotoxicity. The advantage of an in vivo comet assay is that it can be applied to a variety of organs. In general, the liver and stomach are used in an in vivo comet assay. However, there have also been some reports on the in vivo comet assay of the thyroid gland, which is a common target organ for carcinogens in rodents. Ethyl methanesulfonate (EMS) is listed as a compound likely to be the positive control compound of choice in an in vivo comet assay, since it is known to cause DNA damage in a variety of tissues, including the liver, stomach, kidneys, and bone marrow. In contrast, there have been no reports on in vivo comet assays of the thyroid gland in rats treated with EMS. In the present study, we examined whether EMS also exhibits DNA damage in the in vivo comet assay of the thyroid gland in rats. EMS was administered orally at a dose of 200 mg/kg. The results showed a significant increase in mean % tail DNA in the thyroid gland, the extent of which was similar to that in the liver. Since histopathological examination of the thyroid gland revealed no histological findings, the increase in mean % tail DNA was most likely due to DNA damage rather than tissue damage. These results suggest that EMS can be used as a positive control compound in the in vivo comet assay of the thyroid gland in rats.