The specificity of polymerase chain reaction monoclonality in the diagnosis of gastric lymphoma was prospectively evaluated. Gastric mucosal tissue from normal gastric mucosa (N = 13), benign gastric ulcers (N = 3), chronic Helicobacter pylori gastritis (N = 3), gastric mucosa-associated lymphoid tissue (N = 16), and gastric lymphoma (N = 15) was obtained. Polymerase chain reaction amplification of the heavy-chain framework 2A gene was performed. The sensitivity and specificity of heavy-chain clonality, in the detection of gastric lymphoma, were 73.3% and 45.7%, respectively. Determination of monoclonality by polymerase chain reaction methodology is not an acceptable technique for confirming the diagnosis of gastric lymphoma as it is too sensitive, detecting minute populations of clonal lymphocytes that occur in benign diseases as well as larger populations of clonal lymphocytes associated with malignant gastric lymphoproliferative diseases. Southern blot gene rearrangement testing should be utilized to determine clonality in the evaluation of gastric lymphocytic infiltrates.