In this study, we evaluated two biomolecular techniques for discriminating between strains of Escherichia coli isolated form a variety of sources. The DNA of 211 strains of E. coli collected from dairy farms, calves, feces, pigs, primates, humans, and food products was analyzed by pulsed-field gel electrophoresis (PFGE) and repetitive-element polymerase chain reaction using the BOXA1 primer (BOX-PCR). Objectives of the present study were to compare PFGE and BOX-PCR for discriminating among strains of E. coli and investigate their capability in clustering E. coli strains according to the origin of bacterial isolation. Our results showed that PFGE and BOX-PCR were both able to distinguish closely related strains of E. coli; however, PFGE was able to discriminate between isolates indistinguishable by BOX-PCR and interpretation of PFGE data was easier. BOX-PCR proved to have good discrimination power, was less expensive, and could be performed in a PCR thermocycler. Neither of the methods used were effective in clustering E. coli strains according to the source of the organism.