Aims: To develop and evaluate a multiplex PCR(mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli(STEC) and their main virulence marker genes. Methods and Results: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2(stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin(ehlyA). Moreover, two additional genes(rfb O157 and fliC H7), providing the genotypic identification of the O157: H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157: H7 serotype (six isolates) and O157: NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. Conclusions: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. Significance and Impact of the Study: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157: H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.