The aim was to perform a pentavalent PCR assay for the detection of putative virulence genes encoded in VTEC plasmids, katP, espP, subA, stcE, and ehxA. The five-specific primer pairs used in the assay do not interfere with each other and generate amplification products of 914, 774, 556, 399, and 262 bp. It was selected at random 39 strains belonged to 20 serotypes in order to evaluate the multiplex in a wide variety of strains. The results of this study indicate that it is possible to perform simultaneous amplification and search for recognized plasmid-encoded virulence markers from different E. coli serotypes and apply this technique to the genetic characterization of E. coli strains isolated from reservoirs, foods or patients. This complementary technique is a useful tool to detect interstrain differences for epidemiological studies and to provide information that could be related to the risk of human infection.