Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx2 subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx2 subtype in the ovine isolates was shown to be stx2d-Ount (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H−/H8/H40, O91:H−, O123:H−, O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H− possessed a stx2d-O111/OX3a subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx2d (stx2d-Ount/O111/OX3a) subtype. These studies suggest that a specific stx2 subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.