A cancer bioassay on hexavalent chromium Cr(VI) in drinking water reported increased incidences of duodenal tumors in B6C3F1 mice at exposures of 30–180 ppm, and oral cavity tumors in F344 rats at 180 ppm. A subsequent transgenic rodent (TGR) in vivo mutation assay in Big Blue® TgF344 rats found that exposure to 180 ppm Cr(VI) in drinking water for 28 days did not increase cII transgene mutant frequency (MF) in the oral cavity (Thompson et al., 2015). Herein, we extend our analysis to the duodenum of these same TgF344 rats. At study termination, duodenum chromium levels were below either the limit of detection or quantification in control rats, but were 24.6 ± 3.8 μg/g in Cr(VI)-treated rats. The MF in control (23.2 × 10^− 6) and Cr(VI)-treated rats (22.7 × 10^− 6) were nearly identical. In contrast, the MF in the duodenum of rats exposed to 1-ethyl-1-nitrosourea for six days (study days 1, 2, 3, 12, 19, 26) increased 24-fold to 557 × 10^− 6. These findings indicate that mutagenicity is unlikely an early initiating event in Cr(VI)-induced intestinal carcinogenesis.