Despite similar overall prevalences of E. coli O26 and O157 in Scottish cattle, human cases of shiga toxin-producing E. coli O26 infection are usually uncommon and clinically less severe. This discrepancy was investigated by genotyping cattle and human E. coli O26 isolates from Scotland and Europe. The genetic background of some Scottish strains was closely related to strains causing severe infections in Europe. Nonmetric multidimensional scaling found an association between hemolytic uremic syndrome (HUS) and multilocus sequence type ST21 strains and confirmed the importance of stx2 for severe human disease. Although the overall prevalence of E. coli O26 on Scottish cattle farms is equivalent to E. coli O157, prevalence of more virulent strains (stx2+ eae+) is low, reducing human infection risk. However, new data on E. coli O26-associated HUS cases in humans highlights the need for surveillance of non-O157 STEC and the importance and limiting factors for Stx2 phage acquisition.