We developed a polymerase chain reaction (PCR) assay to detect Helicobacter pylori in gastric and/or gastroesophageal biopsy specimens of adults with dyspepsia, compared the method with immunohistochemical analysis and CLOtest (Ballard Medical Products, Draper, UT), and correlated the results of each test with the histologic features of infection. H pylori was identified in 36 (60%) of 60 patients irrespective of biopsy site and testing method. In the gastric biopsy specimens, PCR detected H pylori in 29 (52%) of 56 cases, including 11 (100%) of 11 immunohistochemically and/or CLOtest-positive cases. PCR-positive gastric biopsy specimens correlated with a higher average cumulative inflammatory score compared with PCR-negative specimens (P = .001). In gastroesophageal biopsy specimens, PCR detected H pylori in 15 (34%) of 44 cases, including 1 (20%) of 5 immunohistochemically positive specimens. PCR-positive gastroesophageal junction biopsy specimens did not correlate with a higher average cumulative inflammatory score. Overall, PCR detected an additional 23 cases negative by immunohistochemical analysis and/or CLOtest. This PCR assay identified a significant number of H pylori infections that would not be detected by immunohistochemical analysis and/or CLOtest.