Background. Persistent Helicobacter pylori infection may induce several upper gastrointestinal diseases. Two major virulence factors of H. pylori, vacuolating cytotoxin A (VacA) and cytotoxin-associated gene A (CagA), are thought to be associated with the severity of disease progression. The distribution of vacA and cag-pathogenicity island (cag-PAI) alleles varies in H. pylori isolated from patients in different geographic regions. Aim. To assess the association between mixed infection of H. pylori clinical isolates from Taiwanese patients and the severity of gastrointestinal diseases. Methods. A total of 70 patients were enrolled in this study. Six distinct and well-separated colonies were isolated from each patient and 420 colonies were analyzed to determine the genotypes of virulence genes. Results. The prevalence of mixed infections of all H. pylori-infected patients was 28.6% (20/70). The rate of mixed infections in patients with duodenal ulcer (47.6%) was much higher than that with other gastrointestinal diseases (P < 0.05). Conclusions. H. pylori mixed infections show high genetic diversity that may enhance bacterial adaptation to the hostile environment of the stomach and contribute to disease development.