Colombia, South America has one of the world's highest burdens of infection and gastric cancer. While multidrug antibiotic regimens can effectively eradicate , treatment efficacy is being jeopardized by the emergence of antibiotic-resistant strains. Moreover, the spectrum of and genetic mechanisms for antibiotic resistance in Colombia is underreported. In this study, 28 strains isolated from gastric biopsy specimens from a high-gastric-cancer-risk (HGCR) population living in the Andes Mountains in Túquerres, Colombia and 31 strains from a low-gastric-cancer-risk (LGCR) population residing on the Pacific coast in Tumaco, Colombia were subjected to antibiotic susceptibility testing for amoxicillin, clarithromycin, levofloxacin, metronidazole, rifampin, and tetracycline. Resistance-associated genes were amplified by PCR for all isolates, and 29 isolates were whole-genome sequenced (WGS). No strains were resistant to amoxicillin, clarithromycin, or rifampin. One strain was resistant to tetracycline and had an A926G mutation in its 16S rRNA gene. Levofloxacin resistance was observed in 12/59 isolates and was significantly associated with N87I/K and/or D91G/Y mutations in Most isolates were resistant to metronidazole; this resistance was significantly higher in the LGCR (31/31) group compared to the HGCR (24/28) group. Truncations in and were present in nearly all metronidazole-resistant strains. There was no association between phylogenetic relationship and resistance profiles based on WGS analysis. Our results indicate isolates from Colombians exhibit multidrug antibiotic resistance. Continued surveillance of antibiotic resistance in Colombia is warranted in order to establish appropriate eradication treatment regimens for this population.