AbstractCBX3 is an isoform of the heterochromatin protein 1 family, which is involved in carcinogenesis and promotes the progression of certain types of cancer. The expression level and clinicopathological significances of CBX3 in colorectal cancer (CRC) are still not well reported. In this study, we examined CBX3 protein expression in formalin‐fixed and paraffin‐embedded normal mucosae, hyperplastic polyps, low‐and high‐grade adenomas, and CRC tissue samples using immunohistochemistry. The associations of CBX3 expression levels with clinicopathological parameters, mismatch repair (MMR) protein expression, and kirsten rat sarcoma viral oncogene homolog (KRAS) and B‐raf proto‐oncogene (BRAF) mutations were analyzed. Our results showed that CBX3 protein was negatively expressed in normal mucosae and hyperplastic polyps, as well as in most low‐grade adenomas. Interestingly, CBX3 protein was positively expressed in most high‐grade adenomas and CRC tissues. CBX3 expression level was associated with tumor differentiation (p = 0.012), lymph node metastasis (p = 0.024), TNM stage (p = 0.008) and survival (p = 0.029). CBX3 expression was associated with MMR protein expression (p = 0.011) and KRAS mutation (p = 0.013), but not with BRAF mutation (p = 0.097). Our data suggest that CBX3 may be used as a molecular marker in CRC to evaluate tumor differentiation, lymph node metastasis, and pathological stage.