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Infection with Helicobacter pylori ( H. pylori ) is very common and affecting about 50% of the worldwide population. Several genetic variations have been implicated in determining the clinical susceptibility to this infection. In the current study, we examined the association between C1236T (rs1045642) and C3435T (rs1045642) single nucleotide polymorphisms (SNPs) in the ABCB1 gene and the prevalence of H. pylori infection among Jordanians. A total of 412 subjects (257 H. pylori -positive cases and 155 H. pylori -negative controls) were recruited and participated in the study, and the genotyping of the ABCB1 gene was performed using RFLP-PCR techniques. A significant association was detected between C1236T and H. pylori infection ( p < 0.01). The frequency of CT genotype was significantly higher in the positive cases (40.1%) compared to the controls (21.3%). In addition, the C3435T SNP was weakly associated with H. pylori infection ( p = 0.077). Haplotype analysis of C1236T and C3435T SNPs showed that the TT haplotype was present in 22.7% of the positive cases compared to 30.7% of the negative controls ( p < 0.05, odds ratio = 0.663, 95% CI: (0.483-0.911)). Consequently, the TT haplotype seems to decrease the risk of H. pylori infection. In conclusion, the current results suggest an association between ABCB1 SNPs and H. pylori infection in the Jordanian population.