ESBL producing Escherichia coli (E. coli) have spread in the hospital settings. The aims of this study determination of genetic relationship between Environmental E. coli with PFGE typing and investigation of IS element in blaCTX-M gene of these isolates. A total of 50 E. coli isolates were collected from hospital environmental. The blaCTX-M producing E. coli and IS element of this gene with phylogenetic typing were detected by PCR. The PFGE was performed to detect genetic relationships between this strains. Most of the isolates were from urology wards, other samples were isolated from ICU, surgery and orthopedic ward. The majority of isolates were resistant to cefotaxime and ceftazidime antibiotics and also phosphomycin antibiotic resistant were detected in 10% of isolates. CTX-M gene was detected in 72% of isolates. Moreover, ISEcp1, IS26a, and IS26b were detected upstream of CTX-M in 24%, 8% and 16 of isolates. A phylogroup was the most frequent and PFGE analysis exhibited a diverse distribution of E. coli isolates. The results demonstrated the existence of CTX-M-producing E. coli in a hospital environment which is a source for drug-resistant strains. In the most of strains, ISEcp1 was located in the upstream of CTX-M gene and Orf477 was found in the downstream. However, in some strains, IS26 was inserted within the ISEcp1element. Our results show that despite the fact that antibiotics of phosphomycin are not used in this hospital, resistance to phosphomycin was observed in the environmental E. coli. Copyright © 2021 Elsevier Ltd. All rights reserved.