stx2 genes from 138 Shiga toxin-producing Escherichia coli (STEC) isolates, of which 127 were of bovine origin (58 serotypes) and 11 of human origin (one serotype; O113:H21), were subtyped. The bovine STEC isolates from Australian cattle carried ehxA and/or eaeA and predominantly possessed stx2-EDL933 (103 of 127; 81.1%) either in combination with stx2vhb (32 of 127; 25.2%) or on its own (52 of 127; 40.4%). Of 22 (90.9%) bovine isolates of serotype O113:H21, a serotype increasingly recovered from patients with hemolytic uremic syndrome (HUS) or hemorrhagic colitis, 20 contained both stx2-EDL933 and stx2vhb; 2 isolates contained stx2vhb only. Although 7 of 11 (63.6%) human O113:H21 isolates associated with diarrhea possessed stx2-EDL933, the remaining 4 isolates possessed a combination of stx2-EDL933 and stx2vhb. Three of the four were from separate sporadic cases of HUS, and one was from an unknown source.