Clostridium difficile infection (CDI) is emerging as a cause of diarrhea in infants and children in both community and hospital settings. This increasing incidence has been attributed, in part, to the emergence of a hypervirulent strain of C. difficile, increased antibiotic prescriptions, increased awareness of CDI among healthcare providers and emergence of highly sensitive detection methodologies for CDI. It is essential to have accurate laboratory diagnosis of CDI to ensure patients receive appropriate treatment and that correct infection control measures are put in place. Objectives: The aim of this work was to determine the prevalence of toxigenic C. difficle among hospitalized children complaining of antibiotic associated diarrhea (AAD) in Sohag university hospital. Patients and Methods: Stool samples were collected from 80 hospitalized children admitted in pediatrics department, Sohag university hospital receiving antibiotics and complaining of diarrhea. To diagnose C. difficile, three different approaches were undertaken on stool samples: 1) C. difficile isolation on selective medium cycloserine-cefoxitin-fructose agar plates (CCFA) 2) immunoenzymatic detection of toxins A and B (Xpect Clostridium difficile Toxin A/B immunochromatographic assay) 3) multiplex PCR for detection of C.difficile triose phosphate isomerase gene (tpi) and its toxins A (tcdA) and B (tcdB) genes. Results: The study revealed that the prevalence of toxigenic C.difficile was 13.75 % among the participants by multiplex PCR, 11.2 % by CCFA media and 10 % by Xpect immunochromatoghraphic test. In comparison with multiplex PCR results, the sensitivity and specificity of Xpect Toxin A/B immunochromatoghraphic test were 73 % and 100 % respectively, and for CCFA culture the sensitivity and specificity were 82 % and 100% respectively. The study revealed that cephalosporins were the most commonly received antibiotic (64%) among Clostridium difficile associated diarrhea (CDAD) patients.   Conclusion: C.difficile is an important cause of nosocomial diarrhea in children in Sohag University Hospital, misuse of antibiotics and prolonged hospital stay are major risk factors for acquiring CDI and judicious use of antibiotics and proper infection control measures could minimize C. difficile transmission.