1,4-Dioxane is a genotoxic carcinogen, and its mutagenic properties were recently observed in the liver of guanine phosphoribosyl transferase (gpt) delta transgenic rats. However, the mechanisms of its genotoxicity remain unclear. We analyzed DNA adduct formation in rat livers following 1,4-dioxane treatment. After administering 1,4-dioxane in drinking water at doses of 0, 20, 200, and 5,000 ppm, liver adduct formation was analyzed by DNA adductome analysis. Adducts in treated rat livers were dose-dependently increased compared with those in the control group. Principal component analysis-discriminant analysis (PCA-DA) clearly revealed two clusters of DNA adducts, associated with 0 ppm and low-dose (20 ppm) 1,4-dioxane-treatment versus middleand high-dose (200, 5,000 ppm)-treated rats. After confirming the intensity of each adduct, three adducts were screened as characteristic of 1,4-dioxane treatment. Two of the three candidates contained thymine or cytidine/uracil moieties. Another candidate was identified as 8-oxo-dG based on mass fragmentation together with high-resolution accurate-mass (HRAM) mass spectrometry data. Oxidative stress responses may partly explain the mechanisms of increased mutations in the liver of gpt delta rats following 1,4-dioxane treatment.