Shiga toxigenic Escherichia coli O157: H7 and O111 are capable of causing severe gastrointestinal disease in humans and animals. The present study aims to detect and characterizes  E. coli O157:H7 and O111 in the milk samples collected from cattle with clinical and subclinical mastitis. Out of 240 examined milk samples, 48 E.coli isolates (20%) obtained clinical mastitic cases, while, 22 (13.75%) out of 160 examined milk samples obtained from subclinical mastitic animals. 22 strains (19.7%) O111, 12 strains (5%) O157: H7, 8 strains (3.33%) O128 and 6 strains (2.5%) O119 were recovered from clinical mastitic cases. While serotyping of E. coli re­ceived from subclinical mastitis revealed, 11 strains (6.87%) O111, 5 strains (3.13%) O157: H7, 4 strains (2.5%) O128 and 2 strains (1.25%) O119. Multiplex PCR using O157 and O111 primers revealed the ability detect all the bacteriologically posi­tive samples. Moreover, the multiplex PCR could detect 7 strains of E. coli O111 and 5 strains of O157 from animals with subclinical mastitis, such samples were proved to be negative for O157 and O111 by bacteriological examination which indicated the ability of the sero-group­ specific multiplex PCR assay to detect a very low concentration by STEC organisms.