Abstract Background: Infection with Helicobacter pylori is recognized as the main risk factor for gastric cancer (GC); the clinical outcome of this infection is variable and partially depends on the virulence of the infective strain. This study characterizes H. pylori virulence genes in patients with diverse gastric lesions, from preneoplasia to GC, from a South American region with high GC mortality rates. Methods: We studied the virulence profiles of H. pylori strains to colonize the antrum of 318 patients with non-atrophic gastritis (NAG), 58 patients with preneoplastic lesions (PN), and 90 with GC from Ibagué, Colombia. The presence of 16S rDNA , the cagA and cagE genes, and the vacA s 1 , s 2 , m 1 , and m 2 alleles were determined by PCR. Results: H. pylori infection was detected in 44% of all patients, 41.2% in NAG, 43.1% in PN and 54.4% of GC patients (p= 0.0813). cagA and cagE genes were significantly more frequent in and GC than in NAG (p= <.0001). The vacA s 1 m 1 haplotype was significantly more frequent in PN (68%) and GC (65.3%) than in NAG (37.4%). The frequency of vacA s 2 m 2 haplotype decreased significantly from NAG (42.7%) to PN (12%) and this to GC (4.1%). A total of 23 different genotypes were identified, with cagA +/ cagE +/ vacA s 1 m 1 (84/205) as the more frequent in PN and GC and cagA -/ cagE -/ vacA s 2 m 2 in NAG (49/205). Conclusions: In the population studied, vacA s 2 m 2 was identified as a significant marker for protection against PN and GC, and genotype cagA +/ cagE +/ vacA s 1 m 1 as a marker for increased GC risk. We also found that patients with PN and GC had a higher frequency of cagA +/ cagE +/ vacA s 1 m 1 H. pylori strains known to be aggressive.