Diarrheagenic Escherichia coli still an important pathogen that cause diarrhea which lead to hospital admissions and death specially in children. In order to identify the common pathotypes of E. coli via investigate different virulence genes. A total of 210 stool samples were collected from children under five years presented with diarrhea from different hospitals and private clinics in Wasit province, Iraq, on the other hand, 40 stool samples were collected from healthy children considered as control group. regarding to culture, biochemical tests and API 20E results 100 isolates were supposed to be E. coli. The DNA were extracted to that 100 isolates from diarrheal cases and for 40 isolates of control, concentration of DNA samples were between (50-360 mg/µl ) and the purity between (1.8-2). All isolates studied for detectionvirulence gene of five Diarrheagenic Escherichia Coli strains based on using multiplex Polymerase Chain Reaction technique, by amplified 13 primer (eaeA, bfpB, aggR, astA, pic, hly, stx1, stx2, invE, ipaH, elt, estIa, estIb), and showed the distribution of the strains and its susceptibility to antibiotics. The most frequent pathotypes was Enteropathogenic E.coli 19/42 (45.3%) ) with 9 typical and 10 atypical, followed by Enteroaggregative E. coli 17/42 (40.5%), Enterotoxigenic E. coli 3/42 (7.1%), Enteroinvasive E. coli 3/42 (7.1% ), and 0/42 (0%) in Shigatoxin producing E .coli and no DEC in all control patients. The highest resistance to antibiotics was (95.2%) to Amoxicillin and Ampicillin, respectively, Sulfa-Trimethoprim 92.9%, followed by 85.7% for Tetracycline and Cephalothin, Ceftriaxone 81% and Cefotaxim "clavulanic acid 71.4%. While the lowest resistance was to Chloramphenicol (19 %), Ciprofloxacin (16.7%), Amikacin (7.1%) and no resistance was detected toward Imipenem. We can conclude in this study, multiplex PCR is a swift, and accurate procedure can be used for Diarrheagenic E.coli identification and isolation successfully of strains.