Since 2007, Kenya has experienced an increase in cholera outbreaks characterized by a high fatality rate. In this study, we characterized 81 Vibrio cholerae isolates from diarrhoeal stool samples in Nyanza, a cholera-endemic lake region of Kenya, for virulence properties, clonality and antibiotic susceptibility. Eighty of these isolates were V. cholerae O1 El Tor variants carrying the classical ctxB gene sequence, while one isolate was V. cholerae non-O1/O139. All of the El Tor variants were of clonal origin, as revealed by PFGE, and were susceptible to ampicillin, tetracycline, ciprofloxacin, fosfomycin, kanamycin and norfloxacin. However, the isolates showed resistance to sulfamethoxazole/trimethoprim and streptomycin, and intermediate resistance to nalidixic acid, chloramphenicol and imipenem. The non-O1/O139 isolate carried the cholix toxin II gene (chxA II) and was susceptible to all antimicrobials tested except ampicillin. We propose that an El Tor variant clone caused the Nyanza cholera outbreak of 2007-2008.