There is strong evidence to suggest that E. coli O157 genetic variants (genotypes) are differentially associated with animal and human hosts. The geographic distribution of these genotypes, particularly between countries with different rates of E. coli O157 related disease, is less well understood. Here, we employed molecular typing methods to investigate the extent of genetic variation between large diverse collections of E. coli O157 isolates from countries representing two geographically separate regions, Australia and the United States. This study demonstrates that Australian E. coli O157 populations largely segregate into groups distinct from those seen in the United States. Comparing these genotypes to previously published studies suggest that Australian isolates may be more closely related to E. coli O157 found largely in U.S. cattle rather than those associated with human disease. In addition, the production of Shiga toxin (an important factor associated with human disease) by Australian E. coli O157 was lower than isolates from the United States. This information provides the Australian Meat Industry and government bodies involved in trade negotiations such as DAFF with evidence to support the notion that E. coli O157 isolates in this country may be of lesser threat to public health than isolates from other countries. Future benefits may also arise from the application of this knowledge in commercial and diagnostic laboratories for improved targeting of isolates associated with increased public health risk.