Twenty-three young adult rhesus monkeys from China were evaluated for the presence of Helicobacter pylori. Gastric body and antral biopsy samples were tested for H. pylori by PCR analysis, culture, rapid urease testing, and histologic evaluation. Serologic testing to detect H. pylori immunoglobulin G (IgG) antibodies was performed by using a commercially available human-based enzyme-linked immunosorbent assay (ELISA) test and an ELISA test which utilized homologous H. pylori antigens and an anti-rhesus IgG conjugate. PCR analysis with H. pylori-specific 26-kDa protein primers detected H. pylori in 21 of the 23 rhesus monkeys (91%). Culture testing identified the organism in 12 of the 23 animals (52%). Rapid urease tests were positive for all animals. H. pylori was diagnosed by histological examination in 11 of 23 monkeys (48%). Of the 21 monkeys positive for H. pylori by PCR, only 3 (14%) had positive results by the commercial ELISA test, yielding a sensitivity of 14%, a specificity of 100%, and an accuracy of 22%. However, 19 of the 21 PCR-positive animals (90%) had positive results by the ELISA test with homologous rhesus H. pylori antigen and anti-monkey conjugate, with predicted index values greater than or equal to 0.7 considered positive and values between 0.5 and 0.7 considered equivocal. This test had a sensitivity of 90%, a specificity of 100%, and an accuracy of 91%. Therefore, the ELISA test with rhesus monkey origin components was more accurate for detecting infected animals than the human-based ELISA.