Background & Aims: Two chemotherapies based on either gemcitabine- or 5-fluorouracil (5-FU) remain standard-of-care for pancreatic ductal adenocarcinomas (PDACs). Patients, however, differ in response and thus many clinical trials for experimental drugs have failed. Therefore, there is an urgent need to establish clinically deployable and rapid biomarkers to decide the best chemotherapeutic regimen for subgroups of patients and prevent toxic side effects.Methods: We profiled keratin 17 (K17) expression, a hallmark biomarker of the basal molecular subtype of PDAC, using immunohistochemistry in two cohorts of formalin-fixed paraffin-embedded PDACs (total n=305). These cases were collected through the Know Your Tumor® initiative and three high-volume treating institutions in the U.S. We performed multivariate predictive analyses and determined the threshold to define positive vs negative test.Results: Patients with advance stage disease, low K17, and treated with 5-FU- chemotherapy had three-times longer survival rates compared to counterparts treated with gemcitabine-based chemotherapy. These results contrast to those found in cases with high K17, where no differences were seen with either regimen. Based on RECIST, 72% of low K17 cases responded to treatment compared to 24% of high K17 cases. Predictive value of K17 was independent of mutation status and clinical variables.Conclusion: Our findings indicate that 5-FU-, rather than gemcitabine- based therapies are more likely to extend survival for patients tumors expressing low rather than high K17. A K17 IHC test rapid test in PDACs has been patented (US11,092,603) and it is currently being tested in prospective phase II clinical trials (NCT04469556, NCT02047474 and NCT03991962). Results from these trials will test the presented findings.Funding: This work was supported by academic enrichment funds of the Department of Pathology at the Renaissance School of Medicine, Stony Brook University, the Pancreatic Cancer Action Network, and Perthera Inc. K.R.S. and L.E.H. received support from a Pancreatic Cancer Action Network Acceleration Network Grant (18–65- SHRO). L.E.H. also received the NCI R01CA274355-01, DP2CA280625-01, R00 CA226342-01, the William Raveis Charitable Fund / Rachleff Innovator of the Damon Runyon Cancer Research Foundation, the AACR Career Development Award to Further Diversity, Equity, and Inclusion in Pancreatic Cancer Research, and the Dr. Ralph and Marian Falk Medical Research Trust, Bank of America, Private Bank.Declaration of Interest: K.R.S. and L.E.H. are consultants for KDx Diagnostics Inc. E.B. is an employee of Perthera and owns stocks in the company. E.F.P. has received compensation as an officer of Perthera, Inc. and owns stock in the company. He also has consulted for Theralink Technologies, Inc. and received compensation as Chair of the Science Advisory Board and owns stock in the company. The additional authors report no conflicts of interest.Ethical Approval: All members of our research team members completed Collaborative Institutional Training Initiative (CITI) human subjects and medical ethics training and all studies were performed in accordance with guidelines and regulations of the Stony Brook Medicine Institutional Review Board (IRB) protocol 94651. Patient consent was waived by the IRB agreements for each participating site because the study was restricted to the analysis of de-identified remnant waste surgical pathology specimens.