In Gwangju, Korea, over the last 4 years, human gastrointestinal infection caused by shiga toxin-producing E. coli (STEC) increased. The aim of this study was to ascertain the genetic relatedness of STEC strains by pulsed-field gel electrophoresis (PFGE), as no data on the molecular epidemiology of STEC in Gwangju has yet been published. The PFGE banding patterns were defined for 62 of the 67 STEC strains isolated from cattle and human. There were 11 clonal types in the 11 STEC strains of cattle origin. Among the 11 STEC strains from asymptomatic person, four O91 strains were 100% similarity in band profiles. In the STEC strains isolated from diarrhea patients, same serogroups were grouped to the same cluster; O111 stains were 89.5% similarity, O157 strains 80%, O26 strains 81.5%, and O103 strains 91% similarity, respectively. In conclusion, this is the first report that a large collection of STEC strains from Korea has been analyzed, and a high degree of diversity was observed among the strains analyzed by this technique. PFGE analysis revealed that the strains isolated from human and cattle were closely related within serotypes, and it was useful for epidemiological analysis of STEC. The importance and usefulness of active laboratory surveillance of STEC such as PFGE should be recommended.