A variety of diarrheagenic E coli (DEC) are responsible for causing diarrhea in children under five years age especially in developing courtiers. The aim of this study was to isolate and bacteriological characterizing of DEC from diarrheic children's stool and using molecular identification of DEC pathotypes for better discrimination and investigate their antibiotic resistance patterns. Total of 200 fresh stools specimens were collected from children with diarrhea in Salah Aldin Hospital in Tikrit city, Iraq. The samples were cultured on selective media such as (MacConkey and EMB). Colonies were identified through biochemical reaction and api 20E system and then antibiotic susceptibility profiles were determined. E. coli were isolated and characterized from 200 diarrheagenic stool positive samples, collected from hospitalized children less than 5 years old. A total of 75(40.5%) samples were yielded positive for growth of E. coli Out of these, 46 genotypically-identified DEC were then subjected to multiplex PCR assay targeting certain virulence factors ( SHIG, bfpA, eae, LT, ST, EA, vt1, vt2, daaE, uidA ) for discrimination of pathotyes. 21/46(45.7%) EPEC with 18 atypical and 3 typical, 11/46(24%) ETEC, 3/46 /(6.5%) EAEC, 2/46 (4.3%) for EIEC was detected, 2/46(4.3%) EHEC was also detected, while no DAEC was detected, Also mix pathotypes were detected, more than one pathotype was observed in a number of samples 7/46(15.2%), 5 (aEPEC + ETEC), 2 (aEPEC + EAEC). Moreover, all pathotypes expressed high resistant to Cefotaxime, ampicillin, Piperacillin, Azitromycin, Amikacin. while little resistance to Imipenem, Meropenem, Ciprofloxacin, Norfloxacin was observed. The study concludes EPEC is the dominant pathotype between DEC pathotypes in our society that causes diarrhea in children, and emphasizes the importance of using mPCR assay for best discrimination.