We characterized 97 non-O157 Shiga toxin (stx)–producing Escherichia coli strains isolated from human patients during 2000–2009 from the national reference laboratory in Switzerland. These strains belonged to 40 O:H serotypes; 4 serotypes (O26:H11/H–, O103:H2, O121:H19, and O145:H28/H–) accounted for 46.4% of the strains. Nonbloody diarrhea was reported by 23.2% of the patients, bloody diarrhea by 56.8%. Hemolytic uremic syndrome developed in 40.0% of patients; serotype O26:H11/H– was most often associated with this syndrome. Forty-five (46.4%) strains carried stx2 genes only, 36 strains (37.1%) carried stx1, and 16 (16.5%) strains carried stx1 and stx2. Genes encoding enterohemolysin and intimin were detected in 75.3% and 70.1% of the strains, respectively. Resistance to >1 antimicrobial agent was present in 25 isolates. High genetic diversity within strains indicates that non-O157 stx–producing E. coli infections in Switzerland most often occurred as single cases.