CMTM6 is a major regulator of PD-L1 expression. Aberrant Wnt pathway signaling occurs in most sporadic colorectal cancers (CRC). However, the significance and correlation of β-catenin, CMTM6, and PD-L1 immunohistochemical expression in CRC is still unknown and need to be further verified. We evaluated the expression levels of β-catenin, CMTM6, PD-L1, and MMR (mismatch repair) proteins by immunohistochemistry in CRC tissue microarray (TMA), and evaluated the association among β-catenin, CMTM6, PD-L1 expression, MMR status, and clinicopathological features in 704 CRC patients. Positive expression of PD-L1 in tumor cells (TC) is associated with more frequent dMMR (mismatch repair deficient) status, CMTM6 expression, right colon, and younger CRC patients. The expression of PD-L1 in tumor-infiltrating immune cells (IC) is associated with a higher frequency of adenocarcinoma, β-catenin, and CMTM6 expression. In univariate analysis, age, histological subtype, histologic grade, lymphatic metastasis, TNM stage, MMR status, and expression of PD-L1 protein in IC were significantly associated with the overall survival. In multivariate analysis, age, histologic grade, TNM stage, MMR status, and expression of PD-L1 protein in IC were independent prognostic factors. The overall survival of the adjuvant chemotherapy group was significantly higher than those non-chemotherapy in TNM stage III-IV CRC patients, but no significant overall survival improvement was found in the positive PD-L1 in TC, positive PD-L1 in IC, positive CMTM6, low β-catenin expression, or dMMR status subgroups. Expression of CMTM6 and PD-L1 in CRC are positively associated with β-catenin and reliable biomarkers for the prediction of responding to chemotherapy. The expression of β-catenin/CMTM6/PD-L1 and MMR status may be valuable biomarkers for guiding different treatment strategies in CRC patients.