The aims of this study were to investigate the cytotoxin-associated gene A (cagA) and vacuolating cytotoxin gene A (vacA) subtype in Taiwanese H. pylori isolates from patients with gastroduodenal diseases and to assess the relationship between genotypes of isolates and clinical features. The vacA s1a allele was found in all isolates and vacA m1 allele was found in 15% of isolates. The cagA gene was found in 82.5% of isolates. The vacA s1a/m2 strains had a significantly higher prevalence rate than vacA s1a/m1 strains in Taiwan (p < 0.05). By aligning and comparing the nucleotide and amino acid sequences of vacA from the Taiwanese isolates, the signal sequence and N-terminal region were found to be highly conserved, but the middle region was found to be highly heterogeneous. Determining the relationship between the genotypes and clinical features, we found that the cagA gene was more closely associated with duodenal ulcer than with gastric ulcer and the vacA s1a/m2 strain was more closely associated with active chronic gastritis and atrophic gastritis than with chronic gastritis. Together, our results indicated that (i) the middle region of vacA gene in Taiwanese isolates was heterogeneous; (ii) s1a/m2 vacA strains had a high prevalence in Taiwanese peptic ulcers; and (iii) the cagA gene was significantly associated with duodenal ulcer.