The aim of the study was to evaluate the prevalence of main virulence genes in Helicobacter pylori strains from 116 patients with peptic ulcers (41 cases) and nonulcer diseases (75) by polymerase chain reaction (PCR) with pure cultures and to compare the results with those by multiplex PCR in 39 H. pylori-positive gastric biopsies in another center in Sofia, Bulgaria. Strain susceptibility to amoxicillin, metronidazole, and clarithromycin was determined by agar dilution method. By PCR with pure cultures, coinfections with multiple H. pylori strains were found in 8 (6.9%) patients who were excluded from the statistical analysis. Prevalence of toxigenic type vacA s1 was higher (91.7%) than that usually reported in Europe. cagA-positive genotype was detected in most (81.5%) strains, and almost all of them harbored vacA s1 genotype. Strains with cagA+/vacA s1a genotype were more common (80.6%) than the other genotypes (19.4%, P = 0.0001). The ulcer patients had more often virulent strains than the other patients (92.3% versus 75.4% for cagA+, 100.0% versus 87.0% for vacA s1, 100% versus 84.0% for vacA s1a, and 92.3% versus 73.9% for cagA+/vacA s1a, respectively). The prevalence of H. pylori virulence-associated genes was not associated with patients' sex and age or with the antibacterial resistance of strains. The most common H. pylori genotype was cagA+/vacA s1a. Similar prevalence of cagA-positive (82.1%), vacA s1 (97.4%), and cagA+/vacA s1 strains (79.5%) was found by multiplex PCR in gastric biopsies in the 2nd center. In conclusion, H. pylori strains with virulent genotypes are widespread in symptomatic Bulgarian patients.