Aims: The genetic differences of enterohaemorrhagic Escherichia coli O157 (O157) strains isolated from humans in three widely-separated areas in Japan were analysed to provide information on possible geographic aspects of O157 pathogenicity. Methods and Results: Epidemiologically unlinked O157 strains were isolated in Chiba (300 strains), Fukuoka (260 strains) and Yamagata (81 strains) prefectures. These strains were classified in clades by single nucleotide polymorphism in seven loci and lineage-specific polymorphism assay-6, and differences between the strains in each clade were compared by population genetic analyses using the IS-printing system. Analysis of the clades from the three areas showed linkage disequilibrium of the strains in each clade. Comparison of the genetic differences of strains from the three areas in each clade, from calculated Phi(PT) values, indicated that the strains in each clade were the same population in all three areas, except possibly the clade 12 strains. Conclusions: Population genetics analyses confirmed that the distribution of O157 strains in the clades isolated in three areas in Japan were similar and stable. Significance and Impact of the Study: The pathogenicity of O157 strains infecting humans was comparable due to the similar, stable geographic distribution of O157 clades.