PURPOSE: Helicobacter pylori infection by virulent strains is associated with gastric adenocarcinoma. We aimed to determine whether infection with virulent H. pylori preceded precancerous gastric hypochlorhydria and atrophy in gastric cancer relatives and quantify the extent of virulence factor evolution. EXPERIMENTAL DESIGN: H. pylori strains from 51 Scottish gastric cancer relatives were characterized by genetic fingerprinting and typing the vacuolating cytotoxin gene (vacA), the cytotoxin-associated gene (cagA), and housekeeping genes. We phenotyped strains by coculture with gastric epithelial cells and assessing vacuolation (microscopy), CagA tyrosine phosphorylation (immunoblot), and interleukin-8 secretion (ELISA). RESULTS: Toxigenic (vacA type s1/m1) H. pylori was associated with precancerous gastric hypochlorhydria (P<0.01). Adult family members with this type of H. pylori had the same strain as currently noncohabiting adult family members in 68% cases, implying acquisition during childhood from each other or a common source. We analyzed different isolates of the same strain within families and showed that H. pylori commonly microevolved to change virulence: this occurred in 22% individuals and a striking 44% cases where the strain was shared within families. Microevolution in vacA occurred by extragenomic recombination and in cagA by this or duplication/deletion. Microevolution led to phenotypic changes in virulence. Passage of microevolved strains could be tracked within families. CONCLUSIONS: Toxigenic H. pylori infection precedes and so likely causes gastric hypochlorhydria, suggesting that virulent H. pylori increases cancer risk by causing this condition. Microevolution of virulence genes is common within families of gastric cancer patients and changes H. pylori virulence.