The objective of the current study was to detect the clinical signs of colibacillosis in buffalo calves, isolate E. coli O157:H7, detect its virulence gene eaeA using PCR and estimate its prevalence.The current study sampled 120 buffalo calves aged 1day to 5 months from the Al- Basra veterinary Hospital and Private veterinary clinic within the Basra province between October 2017 and July 2018. A total of 100 calves were naturally diarrheic and the other 20 calves served as controls. The clinical sings in the diarrheic subjects included a significant increase in body temperature, heart rates, respiration rates and capillary refill time as compared to control group. Other clinical signs included whitish to yellowish watery diarrhea with tincture of blood, anorexia, weakness, depression, weak suck reflex, dry oral mucous membranes, cold extremities, weak peripheral pulse, dehydration and death. Using phenotypic characterization tools like MacConky agar, EMB agar, biochemical tests and Viteck, 83 out 100 diarrhea samples confirmed E. coli. Using CT- SMACT agar, 31 out of 83 E. coli isolates were E. coli O157:H7 positive. The PCR result indicated that 47 out of the 83 isolated E. coli samples were positive for eaeA virulence gene. In conclusion, this study is a debut in the report of E. coli and E. coli O157:H7 isolation and genes identification in buffalo calves in Iraq. Therefore, proper prevention and control measures are requisite to curtail the mortality and morbidity rate caused by Colibacillosis.