Nineveh is one of the main governorates of Iraq for sheep ranching. However, in recent years, E.coli has caused many problems and diseases in sheep. Therefore, the current study was conducted to isolate E.coli from sheep and their surrounding environment in two types of breeding systems (open and closed) and to study the effect of the breeding system on the rates of its isolation. During the period from 1 Feb to 30 Apr 2022, 380 samples from milk, skin swabs, oral swabs from lambs, water, dry fodder, feces, bedding and green grass were collected. Eight flocks of sheep classified into two groups open and closed breeding system. Standard microbiological methods were done using Eosin Methylene Blue (EMB), MacConkey and Brilliance agars cultures. Then, the isolates were confirmed using biochemical tests. Molecular confirmation of E.coli was performed using specific primers uidA gene. The current study showed that out of 240 samples, 130 samples from open breeding system at a rate of 54.2% and out of 140 samples, 90 samples from closed breeding system at a rate of 64.3%, were positive for the bacterial isolation and PCR assay. The high isolation rate of E.coli from the sheep closed breeding system, indicates the lack of hygiene procedures application with regard to flocks health, as well as the sanitary procedures followed in animal housing. Close monitoring of food animal production is essential and that it could reduce the potential public health risks in both animal and human medicines.