AIM: A new classification of combined hepatocellular cholangiocarcinoma (CHC) was recently reported. Cancer stem cells have been associated with CHC carcinogenesis. This study examined the association of cancer stem cell marker expression and prognosis in CHC classified using the new classification. METHODS: We enrolled 26 CHC patients and classified them according to the new classification. We evaluated the expression of cancer stem cell markers (CD56, CD133, and epithelial cell adhesion molecule [EpCAM]) by immunohistochemical staining in each component. We analyzed the association between expressions and prognosis. RESULTS: Seven cases were hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) (cHCC-CCA), 12 were HCC and intermediate cell carcinoma (HCC-INT), and seven were intermediate cell carcinoma (INT). The CD133-positive rate tended to be higher in the CCA (42.9%) and INT component (50.0%) than the HCC component (14.3%) in cHCC-CCA. In HCC-INT, the CD133-positive rate in the INT component (83.3%) was significantly higher than the HCC component (8.3%; P = 0.001). For EpCAM, the positive rate in the CCA component (71.4%) and INT component (50.0%) tended to be higher than the HCC component (14.3%) in cHCC-CCA. Overall survival and disease-free survival were significantly worse in cases with CD133-positive (P = 0.048 and P = 0.048, respectively) or EpCAM-positive (P = 0.041 and P = 0.041, respectively) CCA component in cHCC-CCA. CONCLUSIONS: INT and CCA components showed higher expression rates of cancer stem cell markers than the HCC component. CD133 or EpCAM expression in the CCA component was associated with poor prognosis in cHCC-CCA.