Background: Pancreatic cancer is one of the most deadly malignancies with insufficient therapeutic options and poor outcome. Cancer stem cells (CSCs) are thought to be responsible for progression and therapy resistance. We investigated the potential of pancreatic cell lines for CSC research by analyzing to what extent they contain CSC populations and how representative these are compared to clinical tissue. Methods: Six pancreatic cancer cell lines were analyzed by flow cytometry for CD326, CD133, CD44, CD24, CXCR4 and ABCG2. Subsequently, 70 primary pancreatic tissues were evaluated for CD326, CD133 and CD44 by immunohistochemistry. Results: All the cell lines but one showed a stable expression pattern throughout biological replicates. Marker expression in clinical tissue of CD44 distinguished normal patients from pancreatic carcinoma patients with a sensitivity of 50% at 80% specificity and metastasized from nonmetastasized carcinomas with 69% sensitivity at 100% specificity. Conclusions: Our results indicate a link between elevated CD44 expression, malignancy and metastasis of pancreatic tissue. Furthermore, individual pancreatic cell lines show a substantial amount of cells with CSC properties which is comparable with interpatient variability detected in primary tissue. These pancreatic cancer cell lines could thus serve for urgently needed pharmacological CSC in vitro research.