Abstract Background Helicobacter pylori is a human gastric carcinogen that is highly prevalent in Latin American. The prophages of H . pylori show a structured population and contribute to the diversity of this bacterium. However, H . pylori prophages present in American strains have not been described to date. In this study, we identified, characterized, and present the phylogenetic analysis of the prophages present in Colombian H . pylori strains. Methods To characterize Colombian H . pylori strains and their prophages, a Multilocus Sequences Typing (MLST) and a Prophage Sequences Typing (PST), using the integrase and holin genes, were performed. Furthermore, five Colombian H . pylori had their full genome sequenced, and six Colombian H . pylori retrieved from databases, allowing to determine the prophage complete genome and insertion site. Results The integrase gene frequency was 12.6% (27/213), while both integrase and holin genes were present in 4.2% (9/213) of the samples analyzed. The PST analysis showed that Colombian prophages belong to different populations, including hpSWEurope, hpNEurope, hpAfrica1, and a new population, named hpColombia. The MLST analysis classified most of the Colombia strains in the hpEurope population. Conclusions The new H . pylori prophage population revealed that Colombian prophages follow a unique evolutionary trajectory, contributing to bacterial diversity. The global H . pylori prophage phylogeny highlighted five phylogenetic groups, one more than previously reported. After the arrival of Europeans, the Colombian H . pylori bacteria and their prophages formed an independent evolutionary line to adapt to the new environment and new human hosts.