Pancreatobiliary tract strictures result either from malignancies of the biliary tract and pancreas or from nonmalignant etiopathogenesis. The goal of this study was to determine whether KRAS mutations could be identified in residual pancreatobiliary stricture brushings and to compare the performance characteristics of KRAS mutation analysis to cytology and fluorescence in situ hybridization (FISH) for the detection of carcinoma. Residual brushing cytology cell pellets were retrieved from 132 patients with subsequent clinicopathologic follow-up of cholangiocarcinoma (n = 41), pancreatic adenocarcinoma (n = 35), gallbladder cancer (n = 2), and nonmalignant strictures (n = 54). All specimens had a prior cytology and FISH UroVysion results as part of clinical practice. KRAS mutation analysis was performed using the quantitative PCR DxS KRAS Mutation Test Kit. KRAS mutation analysis was successful in 130 of 132 specimens. KRAS mutations and polysomic (ie, positive) FISH results were identified in 24 (69%) and 22 (63%) pancreatic adenocarcinoma specimens, respectively, with a combined sensitivity of 86% (30/35). KRAS mutations and polysomic FISH results were identified in 12 (29%) and 17 (41%) cholangiocarcinoma specimens, with a combined sensitivity of 54% (22/41). KRAS mutations were identified in two patients with primary sclerosing cholangitis, and benign follow-up. Residual cytology specimens can be used to detect KRAS mutations by quantitative PCR. Combined KRAS mutation and FISH analysis appear to increase the cancer detection rate in patients with pancreatobiliary strictures.