Objectives: Widespread use of β-lactam antibiotics could cause resistance to this group of antibiotics in pathogenic bacteria through the production of the enzyme β-lactamases. The aim of this study is to determine the molecular detection of AmpC β-lactamases among clinical Escherichia coli isolated from Ilam hospitals in Ilam, Iran. Methods: One hundred and twelve clinical isolates of E. coli were collected from hospitalized patients and were identified by biochemical tests. They were evaluated for extended spectrum beta-lactamases (ESBLs) production, and the positive strains were subjected to AmpC enzymes; for detection of AmpC cluster genes, multiplex polymerase chain reaction was applied. Results: The analysis showed 62.5% of isolates were ESBLs positive and that five strains revealed the AmpC cluster genes. This is the first report of FOXM cluster genes in E. coli in Iran. Conclusion Based on our results, the prevalence of AmpC β-lactamases is increasing in Iran, which caused failure in antibiotic therapy. So, the current study recommended the revision of antibiotic policy in Iranian hospitals.