Background and objective: Extended Spectrum Beta lactamases (ESBL) such as TEM (Temoneria) is one of the bases of antibiotic resistance in Escherichia coli isolates. The aim of this study was evaluation of TEM Extended Spectrum Beta lactamase producing E. coli, in clinical samples isolated from Zanjan hospitals, by phenotypic and PCR methods. Materials and Methods: In this cross-sectional study 200 E.coli isolates were collected from the clinical specimens such as wound, blood, secretion, urine, and stool from Zanjan hospitals from 2011 to 2012. After identification of isolates by biochemical tests, the antibiotic susceptibility test (Kirby-Bauer method) was done according to CLSI advice against 13 antibiotics. The Combined Disk method was thencarried out for detection of ESBLs and thebla TEM gene was determined by a PCR method. Results: In this study, 77.6 % of E. coli isolates were collected from urine samples. The majority of the samples (71.35%) were resistant to Amoxicillin. By contrast, Imipenem was an effective antibiotic (100% susceptibility) against all isolates. From the total of 200 isolates, the extended spectrum beta-lactamase was detected in 66 (33%) of the strains,only about half of whichwere positive for thebla TEM gene. Conclusion: As only about 15% of the isolates were positive forthebla TEM gene,it is likely that other beta-lactamase enzymes cause the antibiotic resistance. Because resistance agents exist on mobile genetic elements, a rapid detection of these strains couldhelpto prevent their distribution.