The objectives of the current study were detection and characterization of the isolated Escherichia coli non O157 using PCR assay. A total of 90 cheese samples high salt soft cheese, Kareish cheese and Tallaga cheese were examined for E. coli non O157 using modified vancomycin- trypticase soy broth and Sorbitol MacConkey agar plates. Serodiagnosis of E. coli non-O157 has been done using slide agglutination test. Toxigenic E. coli non-O157 isolates were detected using PCR assay. Results postulated that the detection rate of non O157 using biochemical technique was 63.33% in Kariesh cheese, 20% in high salt soft cheese cheese, and 33.33% in Tallaga cheese. Ten different serotypes of E. coli non O157 have been distributed as following O1 (4.44%), O18 (1.11%), O20 (1.11%), O25 (1.11%), O26 (3.33%), O125 (1.11%), O126 (1.11%), O127 (1.11%) and untyped E. coli (18.89%). Regarding PCR results, serotypes group (O1) was positive for Stx2 gene, (O1 and O20) were positive for both Stx2 and hly genes, one serotype group O25 was positive for eaeA gene, one serotype group O26 was positive for hly gene while no detection for Stx1and STa genes. From the aforementioned data, attention must be paid to the problems of E. coli non O157 in foods. Consequently, more restriction and preventive measures should be taken in milk herds, milk production and dairy factories in respect to quality control sanitation and health care.