The Virulence characterization of Escherichia coli in clinical and subclinical mastitic milk samples was studied in this study. Bacteriological examination of 292 milk samples collected from Egypt revealed 44 E. coli organisms (15.068 %).The result showed that the E. coli incidence in clinical mastitic milk higher than subclinical mastitic milk. Serotyping E. coli isolates revealed 41 strains were typed (15.06%). Detection of verotoxin activity of E. coli strains showed that 5 strains (6.4%) belonged to O128: K67, O111: K58 and O126: K58 from clinical mastitic milk samples, while 15 strains (7.01 %) were from subclinical  mastitic cases. For detection of shiga toxin type 2 (stx2) and intimin (eaeA) genes by multiplex PCR, the results revealed positive amplification of intimin gene in 4 strains O111 (9.76%), 3 strains of O128:K67 (7.32%), 2 strains of O26:K60, O55:K59 and O119:K69 (4.88%), one strain of O126:K7(2.44%). Simultaneous amplification of shiga toxin type 2 gene and intimin gene in all 4 E. coli serovar O157: H7, while 3 from serovars O111 (7.32%), from serovars O128 and O26:K60 were 2 (4.88%) and one strain of O55:K59 (2.44%) which confirm that multiplex PCR as­say are useful for identification of STEC possessing the eaeA and stx2 genes.