In order to identify Helicobacter in gallstones of Iranian patients with biliary disease, gallstone and bile samples from 33 patients were subjected to rapid urease test, culture and Multiplex PCR using primers based on 16s rRNA and isocitrate dehydrogenase genes for the identification of Helicobacter genus and H. pylori respectively. This PCR was also done on bile samples from 40 autopsied gallbladders with normal pathology (control group). In 18.1% of stone and 12.1% of bile samples, H. pylori DNA was detected using PCR. Rapid urease and culture tests were negative for all samples. The PCR was negative in the control group. In conclusion, H. pylori DNA was detected in stone samples of Iranian patients with gallstones but we are not sure of their viability. To clarify the clinical role of Helicobacter in gallbladder diseases, studies using accurate tests on larger patient and control groups are needed to ascertain whether this microorganism is an innocent bystander or active participant in gallstone formation.