Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to analyze c-Ki-ras codon 12 mutation in 27 fine needle aspiration biopsy (FNAB) specimens of the pancreas and its adjacent organs for the diagnosis of pancreatic adenocarcinoma. C-Ki-ras codon 12 mutation was present in 14 out of 15 cases of pancreatic adenocarcinoma, the positive rate was 93.33% (14/15); whereas no mutation was detected in those obtained from 12 patients with chronic pancreatitis, pancreatic cyst, gallbladder carcinoma, carcinoma of ampulla of Vater and gastric lymphoma. The results of this study verifies the PCR-RFLP technique as a practical, sensitive, rapid and reliable method for the detection of c-Ki-ras codon 12 mutation in the diagnosis of pancreatic adenocarcinoma.