Background and Objectives: Diarrhoea is one of the leading causes of morbidity and mortality among children under 5 years. Study was aimed to evaluate the role of different categories of diarrhoeagenic Escherichia coli (DEC) in children <5 years of age hospitalised for acute/persistent diarrhoea, by multiplex polymerase chain reaction assay for simultaneous detection of five categories of DEC using eight virulent genes and antibiotic susceptibility pattern of DEC. Materials and Methods: Faecal samples from 355 children with and 150 children without diarrhoea were collected from 2011 to 2013 from King George Medical University and Balrampur Hospital of Lucknow district, Uttar Pradesh, India and were tested for enteric pathogens using conventional diagnostic methods and molecular methods. Results: DEC was detected in 152 (42.81%) children with and 14 (9.33%) in children without diarrhoea respectively. The most common pathotype was enteroaggregative E. coli (EAEC) in cases and controls (16.05% and 5.33%), followed by enteropathogenic E. coli (12.67% and 2.66%), enterotoxigenic E. coli (8.73% and 1.33%), enteroinvasive E. coli (5.35%/and nil) in cases and controls respectively, enterohaemorrhagic E. coli was not detected in any of the diarrhoeal samples. DEC isolates showed a low rate of sensitivity to Ampicillin (07.89%), Amoxycillin-Clavulanic acid (06.57%), Trimethoprim/Sulphamethaxazole (20.39%), Nalidixic acid (24.34%,), Ciprofloxacin (34.34%), Ceftriaxone (23.68%)), Chloramphenicol (33.55%) and Cefoxitin (30.26%) while they were more sensitive to Amikacin (65.78% sensitive), Piperacillin/Tazobactam (61.84% sensitive) and Gentamicin (61.84% sensitive). Interpretation and Conclusions: DEC strains are a significant cause of diarrhoea in children. EAEC was the most frequent pathotype in the study. The high level of antimicrobial resistance in our study raises a broader discussion about the indiscriminate use or misuse of antibiotics and the risks of empirical antibiotic therapy in children of a very young age.