Chemotherapy is the only systemic treatment approved for pancreatic ductal adenocarcinoma (PDAC), with a selection of regimens based on patients' performance status and expected efficacy. The establishment of a potent stratification associated with chemotherapeutic efficacy could potentially improve prognosis by tailoring treatments.Concomitant chemosensitivity and genome-wide RNA profiles were performed on preclinical models (primary cell cultures and patient-derived xenografts) derived from patients with PDAC included in the PaCaOmics program (NCT01692873). The RNA-based stratification was tested in a monocentric cohort and validated in a multicentric cohort, both retrospectively collected from resected PDAC samples (67 and 368 patients, respectively). Forty-three (65%) and 203 (55%) patients received adjuvant gemcitabine in the monocentric and the multicentric cohorts, respectively. The relationship between predicted gemcitabine sensitivity and patients' overall survival (OS) and disease-free survival (DFS) were investigated.The GemPred RNA signature was derived from preclinical models, defining gemcitabine sensitive PDAC as GemPred+. Among the patients who received gemcitabine in the test and validation cohorts, the GemPred+ patients had a higher OS than GemPred- (p = 0.046 and p = 0.00216). In both cohorts, the GemPred stratification was not associated with OS among patients that did not receive gemcitabine. Among gemcitabine treated-patients, GemPred+ patients had significantly higher OS than the GemPred-: 91.3 months (95% CI: 61.2-not reached) vs 33 months (95% CI: 24-35.2); HR 0.403 (95% CI: 0.221-0.735, p = 0.00216). The interaction test for gemcitabine and GemPred+ stratification was significant (p = 0.0245). Multivariate analysis in the gemcitabine-treated population retained an independent predictive value.The RNA-based GemPred stratification predicts the benefit of adjuvant gemcitabine in PDAC patients.