Hexavalent chromium (CrVI) has been shown to induce tumors in the rat oral cavity following chronic drinking water exposure at 180 ppm; however the mode of action (MOA) for these tumors is unknown. We conducted a transgenic rodent mutation assay in Big Blue F344 rats, following OECD Technical Guideline 488, to test whether CrVI induces oral tumors by a mutagenic MOA. Tumors in the NTP CrVI cancer bioassay arose from the gingival epithelium surrounding the upper molars. Therefore, we assessed mutation frequency in gingival/palate and gingival/buccal regions of the oral cavity. Because these tissues are not commonly studied in the assay, we optimized collection methods and conducted a positive control study exposing male F344 Big Blue rats to 10 ppm 4-nitroquinoline N-oxide (4NQO), a mutagenic carcinogen in the oral cavity, in drinking water for 28 days. At day 31, 4NQO induced statistically significant increases in mean mutant frequencies in the gingiva/palate (45.2 ± 8.6 x 10^-6 in control vs 2271 ± 458 x 10^-6 in treated) and gingiva/buccal epithelium (51.2 ± 19.6 x 10^-6 in control vs 1091 ± 146 x 10^‑6 in treated). Having established ability to detect mutations in these tissues, we conducted a 28-day dosed water study with 10 ppm 4NQO (positive control) and 180 ppm CrVI. At Day 31, a statistically significant increase in mutant frequency was observed in gingiva/palate region of 4NQO-treated rats (49.8 ± 17.8 x 10^-6 vs 1818 ± 362 x 10^-6), but not CrVI-treated rats (57.8 ± 9.1 x 10^-6). Similarly, a statistically significant increase in mutant frequency was observed in gingiva/buccal region of 4NQO-treated rats (39.1 ± 7.5 x 10^-6 vs 688 ± 250 x 10^-6) but not CrVI-treated rats (44.4 ± 25.1 x 10^-6). These findings support that CrVI does not act by a mutagenic MOA in the rat oral cavity and demonstrate the utility of this assay for MOA research.