The tumor microenvironment, including cancer-associated fibroblasts (CAFs), plays various clinical roles in cancer growth. CAFs are a heterogeneous population and express a variety of mesenchymal markers. However, the clinical roles for CAFs expressing different markers in pancreatic ductal adenocarcinoma (PDAC) remain unknown. We reviewed 67 resected PDAC patients who had not received preoperative therapy. Each primary tumor was analyzed for vimentin and α-smooth muscle actin (α-SMA) expression by immunohistochemical and dual immunofluorescence staining. There was no correlation between the percentage of cells expressing vimentin and α-SMA in the tumor stroma (Pearson's correlation coefficient: r = 0.171). Higher vimentin expression (p = 0.018) was associated with significantly shorter overall survival in PDAC patients. Using dual immunofluorescence staining, vimentin-positive CAFs were divided into two subpopulations: co-expression of α-SMA, and no co-expression of α-SMA. In PDAC, the level of co-expression had no effect on survival using univariate analysis (median survival time, 33.3months for low co-expression vs. 18.2months for high co-expression; log-rank, p = 0.143). However, multivariate analysis clarified that CAFs expressing vimentin alone was an independent predictor of poor survival (p = 0.014; hazard ratio, 2.305; 95% confidence interval, 1.181-4.497). Vimentin-positive CAFs without co-expression of α-SMA were associated with poor survival in PDAC, and CAFs possessed molecular and functional heterogeneity in this disease.