To identify a gemcitabine resistance-associated gene signature for risk stratification and prognosis prediction in pancreatic cancer. Pearson correlation analysis was performed with gemcitabine half maximal inhibitory concentration (IC50) data of 17 primary pancreatic cancer lines from Genomics of Drug Sensitivity in Cancer (GDSC) and the transcriptomic data from GDSC and Broad Institute Cancer Cell Line Encyclopedia, followed by risk stratification, expression evaluation, overall survival (OS) prediction, clinical data validation and nomogram establishment. Our biomarker discovery effort identified a 14-gene signature, most of which featured differential expression. The 14-gene signature was associated with poor OS in E-MTAB-6134 (HR 2.37; 95% CI 1.75-3.2; p < 0.0001), pancreatic cancer-Canada (PACA-CA) (HR 1.76; 95% CI 1.31-2.37; p = 0.00015), and 4 other independent validation cohorts: pancreatic cancer-Australia (PACA-AU) (HR 1.9; 95% CI 1.38-2.61; p < 0.0001), The Cancer Genome Atlas (TCGA) (HR 1.73; 95% CI 1.11-2.69; p = 0.014), GSE85916 (HR 1.97; 95% CI 1.14-3.42; p = 0.014) and GSE62452 (HR 1.82; 95% CI 1.02-3.24; p = 0.039). Multivariate analysis revealed that the 14-gene risk score was an independent pancreatic cancer outcome predictor in E-MTAB-6134 (p < 0.001) and TCGA (p = 0.006). A nomogram including the 14-gene was established for eventual clinical translation. We identified a novel gemcitabine resistance gene signature for risk stratification and robust categorization of pancreatic cancer patients with poor prognosis.