DNA double-strand breaks (DSBs) are considered as one of the primary causes of cancer but their induction by hydrogen peroxide (H2O2) is still controversial. In this work, we studied whether the high levels of H2O2 produced in the thyroid to oxidize iodide could induce DNA modifications. Scores of DNA damage, in terms of strand breaks, were obtained by comet assay (alkaline condition for single-strand breaks (SSBs) and neutral condition for DSBs). We demonstrated that in a rat thyroid cell line (PCCl3), non-lethal concentrations of H2O2 (0.1–0.5 mmol/l) as well as irradiation (1–10 Gy) provoked a large number of SSBs (∼2–3 times control DNA damage values) but also high levels of DSBs (1.2–2.3 times control DNA damage values). We confirmed the generation of DSBs in this cell line and also in human thyroid in primary culture and in pig thyroid slices by measuring phosphorylation of histone H2AX. l-Buthionine-sulfoximine, an agent that depletes cells of glutathione, decreased the threshold to observe H2O2-induced DNA damage. Moreover, we showed that DNA breaks induced by H2O2 were more slowly repaired than those induced by irradiation. In conclusion, H2O2 causes SSBs and DSBs in thyroid cells. DSBs are produced in amounts comparable with those observed after irradiation but with a slower repair. These data support the hypothesis that the generation of H2O2 in thyroid could also play a role in mutagenesis particularly in the case of antioxidant defense deficiency.