There is a growing concern of a public health risk associated with non-O157 Shiga toxin-producing Escherichia coli (STEC) since E. coli serogroups O26, O45, O103, O111, O121, and O145 are frequently implicated in outbreaks of human illness worldwide. Recently, the Food Safety and Inspection Service of the U.S. Department of Agriculture declared these six STEC O groups to be adulterants in beef. We describe here a rapid, sensitive, and highly specific enzyme-linked immunosorbent assay (ELISA) for the detection of these top six non-O157 STEC O groups. The assays were tested against 174 reference E. coli O groups, with 60 clinical isolates belonging to the target O groups and 10 non-E coli strains belonging to the family Enterobacteriaceae. Assays for serogroups O103, O111, and O121 exhibited 100% specificity, while assays for serogroups O26 and O45 had 98.2% specificity, and O145 had 99.1% specificity. ELISA conducted using artificially inoculated ground beef samples displayed 100% accuracy. The sensitivity of the assay was 5×10(5) colony-forming unit (CFU)/mL, with limits of detection in the range of 1-10 CFU/25 g of ground beef sample following enrichment. The findings of the study suggest that the assay described is simple and rapid, and can be employed to detect target STEC O groups in beef and other food samples. In addition, the assay provides a conceptual framework that can be adapted for the development of similar tests for the rapid detection of other serogroups of E. coli.