Gallbladder cancer (GBC) is a rare but highly aggressive cancer for which no well-accepted prognostic biomarkers have been identified. Thymus cell antigen 1 (Thy1), also known as cluster of differentiation (CD)90, and integrin α6 (ITGA6), also known as CD49f, are important molecules in cancer and putative markers of various stem cell types. However, their role in GBC remains to be elucidated. In the present study, Thy1 and ITGA6 expression status in clinical GBC samples, which comprised squamous cell/adenosquamous carcinoma (SC/ASC) and adenocarcinoma (AC) subtypes, was investigated. The associations between Thy1 and ITGA6 expression and clinical parameters and survival rate were analyzed separately. The THY1 and ITGA6 messenger RNA levels were significantly higher in both SC/ASC and AC tissues than in adjacent non-tumor tissues (all P<0.001). These results were subsequently confirmed by immunohistochemical analyses. Overexpression of Thy1 and ITGA6 was correlated with poor differentiation, large tumor size, lymph node metastasis and great invasiveness in SC/ASC (Thy1, P=0.045, P=0.005, P=0.003 and P=0.009, respectively, and ITGA6, P=0.029, P=0.011, P=0.009 and P=0.004, respectively) and AC (Thy1, P=0.027, P<0.001, P=0.003 and P 0.004, respectively, and ITGA6, P=0.002, P=0.003, P=0.006 and P=0.006, respectively). Both Thy1 and ITGA6 were expressed at higher levels in AC with advanced tumor-node-metastasis stage (TNM) than in AC with low TNM stage (P=0.001 and P=0.018, respectively). In addition, patients with elevated Thy1 or ITGA6 expression had shorter overall survival than those with negative Thy1 or ITGA6 expression. Multivariate Cox regression analysis demonstrated that Thy1 (SC/ASC, P=0.001 and AC, P=0.005) and ITGA6 (both P=0.003) were independent predictors of poor prognosis in both SC/ASC and AC patients. In conclusion, Thy1 and ITGA6 could be clinical prognostic markers for GBC.