With the goal of differentiating H. pylori in order to clarify its pathogenicity, we have conducted a restriction endonuclease of the chromosomal DNA of 24 strains of H. pylori employing the restriction endonucleases, NotI, CPOI, KpnI, and SmaI using pulsed-field gel electrophoresis (PFGE). With NotI, these 16 strains out of 24 strains were completely digested, and 16 clearly different electrophoretic patterns of NotI digested chromosomal DNA fragments were seen, permitting us to successfully differentiate H. Pylori. We also estimated the genome size of each strain, ranging from 1326 to 2360 kilobases. Using this method, we confirmed our previously established nude mouse model of infection with H. pylori by demonstrating that the mice were infected with the H. pylori administered in this study. Thus, we have demonstrated the digested with NotI by PFGE is an effective method for differentiating H. pylori and estimating the size of DNA of H. pylori.