Background and objective: Escherichia coli strains are a member of Enterobacteriacea which are commonly isolated from orthopedic wounds. Recently, by ESBL production, E.coli strains are resistant to most common antibiotics and causes strong worldwide problem in treatment. The aim of this study was evaluation of ESBL production among E.coli strains isolated from orthopedic wounds of Firozgar and Akhtar hospitals during 1389-90 by phenotypic and molecular methods.Materials and methods: During 1389-90 (2011), 60 orthopedic wounds were collected from Firozgar and Akhtar hospitals by sterile condition. The E.coli strains were isolated and identified based on bacterial and biochemical standard methods. Their resistant evaluated against common antibiotics by disk diffusion method (CLSI standard). ESBL screening was done by phenotypic DDST/DDT methods. Consequently, DNA was extracted by kit and the frequency of TEM, SHV, PER-1 genes were evaluated by PCR method.Results: Of 60 collected orthopedic wounds from Firozgar and akhtar hospitals, E.coli strains (48.33%) was detected. Their resistant to tested antibiotics by Kirby-Bauer method were, 41.73% to cefpodoxim, 68.96% to cefotaxim, 51.72% to ceftazidim, 48.27% to cephalexin and gentamicin, ciprofloxacin, chloramphenicle, cefepime and imipenem 44.82%, ceftriaxone 41.37% and aztronam 34.48%. By DDST/DDT methods, 20.68% of E.coli strains were ESBL positive. The frequency of SHV, TEM and PER-1 were 33%, 33% and 50%, respectively by PCR method.Conclusion: Based on searches, ESBL production in E.coli strains is mostly related to CTXM, SHV, TEM genes and PER-1 was detected before in Pseudomonas spp, Acinetobacter spp, Proteus spp. The co-existence of PER-1 was reported in 7.5% of Klebsiella strains in Iran. Based on data, this is the first time that PER-1 was detected in E.coli strains without existence of any other ESBL genes. However, the confirmation of this results need more sample size, sequencing and comparison the results by data bank.