Shiga-toxigenic Escherichia coli (STEC) is an important cause of diarrheal disease. The most notorious STEC serotype is O157:H7, which is associated with hemorrhagic colitis and hemolytic-uremic syndrome (HUS). As a result, this serotype is routinely screened for in clinical microbiology laboratories. With the bias toward the identification of the O157 serogroup in routine diagnostic processes, non-O157 STEC has been largely underrepresented in the epidemiology of STEC infections. This diagnostic bias is further complicated by the fact that many non-O157 STEC infections cause nonspecific gastroenteritis symptoms reminiscent of enteric viral infections. In this study, real-time PCR was used to amplify Shiga toxin genetic determinants (stx(1) and stx(2)) from enriched stool samples that were initially submitted for the testing of enteric viruses in patients with suspected viral gastroenteritis between May and September of 2006, 2007, and 2008 (n = 2,702). Samples were submitted from the province of Alberta, Yukon, the Northwest Territories, and Nunavut, Canada. A total of 38 samples (1.4%) tested positive for Shiga toxin genes, and 15 isolates were cultured for further characterization. Several of the serotypes identified (O157:H7, O26:HNM, O26:H11, O103:H25, O121:H19, and O145:HNM) have been previously associated with outbreaks and HUS. This study outlines the importance of combining molecular methods with classical culture techniques to enhance the detection of emerging non-O157 as well as O157 serotypes in diarrheal stool samples. Furthermore, atypical diarrhea disease caused by non-O157 STEC can be routinely missed due to screening only for viral agents.