BACKGROUND: Despite intent to cure surgery with negative resection margins, locoregional recurrence is common in pancreatic cancer. AIMS: To determine whether detection of K-ras gene mutation in the histologically negative surgical margins of pancreatic cancer reflects unrecognised disease. PATIENTS: Seventy patients who underwent curative resection for pancreatic ductal adenocarcinoma were evaluated. METHODS: All patients had surgical resection margins (pancreatic transection and retroperitoneal) that were histologically free of invasive cancer. DNA was extracted from these paraffin embedded surgical margins and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation at codon 12. Detection of K-ras mutation was correlated with standard clinicopathological factors. RESULTS: K-ras mutation was detected in histologically negative surgical margins of 37 of 70 (53%) patients. A significant difference in overall survival was demonstrated between patients with margins that were K-ras mutation positive compared with negative (median 15 v 55 months, respectively; p = 0.0008). By univariate and multivariate analyses, detection of K-ras mutation in the margins was a significant prognostic factor for poor survival (hazard ratio (HR) 2.8 (95% confidence interval (CI) 1.5-5.3), p = 0.0009; and HR 2.8 (95% CI 1.4-5.5), p = 0.004, respectively). CONCLUSIONS: Detection of cells harbouring K-ras mutation in histologically negative surgical margins of pancreatic cancer may represent unrecognised disease and correlates with poor disease outcome. The study demonstrates that molecular-genetic evaluation of surgical resection margins can improve pathological staging and prognostic evaluation of patients with pancreatic ductal adenocarcinoma.