Cholangiocarcinoma is a highly malignant form of gastrointestinal cancer with an unfavorable prognosis. The novel oncogene chromodomain helicase/ATPase DNA binding protein 1-like (CHD1L) has been confirmed to serve a vital role in numerous types of cancer, including liver cancer. Mismatch repair (MMR) is a common DNA repair process that contributes to the preservation of the integrity and stability of genetic substances. Human mutL homolog 1 gene (hMLH1) is an important MMR protein family member. The present study aimed to evaluate the pathological and clinical features of cholangiocarcinoma, and to investigate the clinical significance of CHD1L and hMLH1 expression in cholangiocarcinoma. A total of 108 samples from cholangiocarcinoma tumor tissues and 60 samples from normal bile duct tissue were obtained from patients admitted to The Second Affiliated Hospital of Nanchang University between May 2005 and May 2014. All cholangiocarcinoma cases were pathologically confirmed. The expression of CHD1L and hMLH1 was examined by immunohistochemistry analysis. The expression of CHD1L in cholangiocarcinoma (94.44%) was significantly higher than in normal bile duct tissues (40.00%). CHD1L expression was associated with gallstone history, serum carbohydrate antigen 19-9 (CA19-9) level and Tumor-Node-Metastasis (TNM) stage (P0.05) between the 3-year accumulate survival rates for hMLH1-positive and -negative patients (38.90 and 33.30%, respectively). High CHD1L expression and low hMLH1 expression levels were observed in patients with cholangiocarcinoma, and their abnormal expression patterns were associated with the progression of malignancy and an unfavorable disease prognosis. Therefore, CHD1L and hMLH1 may be potential prognostic biomarkers for cholangiocarcinoma.