Helicobacter pylori (H. pylori) eradication treatment includes a proton pump inhibitor and two antibiotics: amoxicillin and clarithromycin. The goal of that treatment is to eradicate the infection in at least 90% of the patients. Failure to eradicate the infection can have multiple causes, among which is the presence of point mutations in the antimicrobial target genes.To characterize the mutations present in the pbp1a gene and their possible association with resistance to amoxicillin in vitro.Susceptibility to amoxicillin was evaluated in 147 isolates of H. pylori from the Colombian municipality of Túquerres. PCR amplification and sequencing of the glycosyltransferase domain of the pbp1a gene were carried out on Túquerres isolates, and the association between mutations and resistance was evaluated.A total of 5.4% (8/147) Túquerres isolates were resistant to amoxicillin in vitro. PCR amplification of the glycosyltransferase domain of the pbp1A gene was performed on 87.5% of the amoxicillin-resistant isolates in vitro, and in the DNA sequencing analysis, a total of 2 changes of amino acids from 3 DNA mutations that encoded the PBP1A-1 protein were observed.The present study is the first report on pbp1a gene mutations in H. pylori isolates coming from a population in Túquerres. Mutations that have not been reported in previous studies were found.