Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.