Shiga toxin–producing Escherichia coli (STEC) strains are foodborne pathogens that negatively impact human health and compromise food safety. Serogroup O157 is the most frequently isolated and studied STEC serogroup, but six others (O26, O45, O103, O111, O121, and O145) have also been identified as significant sources of human disease and collectively have been referred to as the “top six” pathogenic serogroups. Because detection methods for non-O157 serogroups are not yet refined, the objective of this study was to compare the effectiveness of immunomagnetic separation (IMS) for recovery of serogroup O157 isolates with that for each of the top six E. coli serogroups in pure and mixed cultures of STEC at 103 to 107 CFU/mL. After serogroup-specific IMS, DNA was extracted from cultured isolates to analyze the specificity of each IMS assay using conventional and quantitative PCR. In pure cultures, DNA copy number obtained after IMS was lower for O111 and O157 (P < 0.01) than for other serogroups. Based on quantitative PCR (qPCR) analyses, specificity was reduced for all IMS assays when STEC isolates were mixed at 7 log CFU/mL, although the O157 IMS assays recovered only O157 over a wider range of concentrations than did assays for non-O157 serogroups. At the lowest dilution tested, conventional PCR was specific for all serogroups except O121 and O145. For these two serogroups, no dilution tested recovered only O121 or O145 when evaluated with conventional PCR. Refinements to IMS assays, development of selective media, and determination of optimal enrichment times to reduce background microflora or competition among serogroups would be especially beneficial for recovery of O111, O121, and O145 serogroups to improve STEC detection and isolation.