Objective To establish a detection technique for H.pylori(HP) infection in Mongolian gerbils using nested PCR technique. Methods H. pylori was cultured in vitro and inoculated into Mongolian gerbils. At the 10th week after infection, the HP in the gastric juice of Mongolian gerbil was detected by conventional PCR assay and the gastric juice, gastric mucosa, duodenal contents and colon stool were examined by nested PCR. Rapid urease test and ELISA were used to analyze the accuracy of the nested PCR assay. All of the PCR products were verified by sequencing. Results The positive rate of gastric juice detected by conventional PCR was 30%, while the positive rates of gastric juice, gastric mucosa, duodenal contents and colon stool detected by nested PCR were 100%, 100%, 90%, and 10%, respectively. The positive detection rates of rapid urease test and serum ELISA were 100% and 0%, respectively. Comparing the results of different methods, both the positive rates of gastric juice and gastric mucosa detected by nested PCR and the detection rate of rapid urease test were 100%, but the results of conventional PCR detection of gastric juice, the nested PCR detection result of stool in colon and of serum ELISA assay were lower than other methods. Conclusions Due to its high accuracy and sensitivity, the nested PCR assay of gastric juice can be used for the long-time detection of H.pylori infection in Mongolian gerbils, especially useful in the experiments of prevention and treatment of H. pylori infection.