Oral exposure to high concentrations of hexavalent chromium (Cr(VI)) induces mucosal damage and tumors in the mouse duodenum. Previous microarray-based transcriptomic analyses of frozen homogenized mouse duodenal tissue have demonstrated Cr(VI)-induced alterations in various cellular pathways and processes. According to x-ray fluorescence microscopy, chromium localizes primarily to the duodenal villi following exposure to Cr(VI); therefore, we hypothesized that the transcriptomic response to Cr(VI) varies in the crypt and the villus compartments as a result of differences in exposure as well as cell population. To provide a more complete assessment of transcript responses to Cr(VI), templated oligomer sequencing technology (TempO-Seq) was used to conduct transcriptomic analyses separately on crypt and villus regions from formalin-fixed paraffin-embedded transverse duodenal sections from the same study in which microarray-based analyses were previously conducted. A total of 28 groups (7 doses, 2 timepoints, 2 tissue compartments) were analyzed for differential gene expression in each group compared to controls, dose-response using BMDExpress, and gene set enrichment. Fewer than 21 genes were altered in the crypt compartment of mice exposed to 0.1-5 ppm Cr(VI) for 7 or 90 days, while hundreds to thousands of genes were altered in the crypt at >=20 ppm Cr(VI). Many more genes were altered in the villi than crypts at low concentrations, and increased less drastically with increasing concentration. Notably, although there were more genes altered at low concentrations in the villi, these gene changes did not result in enrichment of gene sets, indicating a non-specific stress response. Consistent with histological evidence for crypt proliferation, a significant dose-dependent increase in genes that regulate mitotic cell cycle was prominent in the crypt, while subtle in the villus, when compared to time-matched controls. Minimal transcriptomic evidence of DNA damage response in either the crypts or the villi is consistent with published in vivo genotoxicity data. These results are relevant to modes of action that have been proposed for Cr(VI)-induced small intestine tumors in mice.