Culture-based methods to detect the six major non-O157 (O26, O45, O103, O111, O121 and O145) Shiga toxin-producing E. coli (STEC) are not well established. Our objectives of this study were to develop a culture-based method to detect the six non-O157 serogroups in cattle feces and compare the detection with a PCR method. Fecal samples (n = 576) were collected in a feedlot from 24 pens during a 12-week period and enriched in E. coli broth at 40° C for 6 h. Enriched samples were subjected to immunomagnetic separation, spread-plated onto a selective chromogenic medium, and initially pooled colonies, and subsequently, single colonies were tested by a multiplex PCR targeting six serogroups and four virulence genes, stx1, stx2, eae, and ehxA (culture method). Fecal suspensions, before and after enrichment, were also tested by a multiplex PCR targeting six serogroups and four virulence genes (PCR method). There was no difference in the proportions of fecal samples that tested positive (74.3 vs. 77.4%) for one or more of the six serogroups by either culture or the PCR method. However, each method detected one or more of the six serogroups in samples that were negative by the other method. Both culture method and PCR indicated that O26, O45, and O103 were the dominant serogroups. Higher proportions (P < 0.05) of fecal samples were positive for O26 (44.4 vs. 22.7%) and O121 (22.9 vs. 2.3%) serogroups by PCR than by the culture method. None of the fecal samples contained more than four serogroups. Only a small proportion of the six serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. The culture method and the PCR method detected all six serogroups in samples negative by the other method, highlighting the importance of subjecting fecal samples to both methods for accurate detection of the six non-O157 STEC in cattle feces.