Healthy cattle are considered the main reservoir of Shiga toxin-producing Escherichia coli (STEC) strains, so in some places in the world, products derived from beef are the most common source for disease outbreaks caused by these bacteria. Therefore, in order to guarantee that the beef produced by our slaughterhouses is safe, there is a need for continuous monitoring of these bacteria. In this study, 215 beef cuts were evaluated, including chilled vacuum-packed striploins (151 samples), rib eyes (30 samples), and knuckles (34 samples), from March to June, 2018. These meat samples were collected from the slaughter of unconfined cattle, being arbitrarily collected from eight meat-processing companies in São Paulo state, Brazil. Each sample was examined for the presence of STEC toxin type ( stx 1 and/or stx 2 genes) and also the E. coli attaching-and-effacing ( eae ) gene, which were determined by a multiplex PCR assay. Here we show that the major seven STEC strains (O serogroups O26, O45, O103, O111, O121, O145, and O157) are not detected in any of the analyzed beef cut samples; however, three of them presented the virulence eae gene. Therefore, the absence of STEC strains in the beef samples may be an indication of the low prevalence of this pathogen in the cattle herd on the farm, associated with good hygiene and handling practices adopted by the meat industry.