Escherichia coli O157:H7 is a foodborne pathogen distinguished from typical E. coli by the production of Shiga toxins (Stx) and the inability to ferment sorbitol (SOR) and to express α-glucuronidase (GUD) activity. An allele-specific probe for the GUD gene (uidA) and multilocus enzyme electrophoresis were used to elucidate stages in the evolutionary emergence of E. coli O157: H7. A point mutation at + 92 in uidA was found only in O157:H7 and its nonmotile relatives, including a SOR+ O157:H⁻ clone implicated in outbreaks of hemolytic-uremic syndrome in Germany. The results support a model in which O157:H7 evolved sequentially from an O55:H7 ancestor, first by acquiring the Stx2 gene and then by diverging into two branches; one became GUD⁻ SOR⁻, resulting in the O157:H7 clone that spread worldwide, and the other lost motility, leading to the O157:H⁻ clone that is an increasing public health problem in Europe.