Enterohemorrhagic Escherichia coli (EHEC) O26 infections cause severe human diseases such as hemolytic uremic syndrome and encephalopathy, and is the predominant serogroup among non-O157 EHEC in many countries. Shiga toxin (Stx), which consists of two distinct types (Stx1 and Stx2), plays a central role in EHEC pathogenesis. The major stx gene type in EHEC O26 strains is stx1, although isolates with only stx2 have emerged in Japan since 2012 and have been reported in Europe. In this study, we selected 27 EHEC O26 strains isolated in Japan and identified a distinct genetic clade within sequence type (ST) 29, designated ST29C1, that carried only stx2 and had the plasmid gene profile ehxA+/katP-/espP+/etpD-. We showed that ST29C1 strains produced higher Stx2a levels, and greater virulence in Vero cells and in germ-free mice than other lineages. We also showed that ST29C1 was a distinct phylogenetic clade by SNP analysis using whole genome sequences and clearly differed from the major European EHEC O26 virulent clone, which was designated ST29C2 in this study. The combination of toxin production analysis, virulence analysis in Vero cells and germ-free mice, and phylogenetic analysis identified a newly emerging virulent EHEC clade.