The treatment for colorectal cancer is largely surgical followed by adjuvant chemotherapy in high-risk cases. In patients with stage II cancer, there is no clear benefit for chemotherapy, and the current tools for assessment of risk are inadequate. A recent study identified that colorectal cancer with a gene signature similar to undifferentiated colonic stem cells was associated with a worse outcome. It was later shown that loss of CDX2 detected by immunohistochemistry (IHC) alone resulted in a worse prognosis and that this could be used to predict patients who would benefit from chemotherapy. Having observed that CDX2 expression can be patchy, we elected to validate these prior results for clinical practice using whole-slide IHC. The pathology of all cases was reviewed, and 3 blocks were selected for CDX2 IHC. We also expanded the panel beyond CDX2 to assess whether other markers in the gene signature including CDX1, Muc2, GPX2, and villin could better predict outcome. Among 210 cases, CDX2 expression was diffusely lost in 11% and focally lost in 23% of cases. There was no difference in survival based on CDX2 expression, but Muc2 loss was associated with reduced survival (hazard ratio, 3.32; 95% confidence interval, 1.20 to 9.20). No significant differences in outcome were identified based on CDX1, GPX2, or villin expression. In keeping with this, assessment of The Cancer Genome Atlas gene expression data demonstrated that decreased Muc2 expression was associated with reduced overall survival. Our results with whole-slide IHC are different from the previous studies and caution against the use of CDX2 in isolation as a prognostic marker in clinical practice. We have identified that loss of Muc2 is associated with reduced survival. This supports the use of the colonic differentiation gene expression signature to identify high-risk patients but cautions against the use of any one IHC-based marker in isolation.