Background: Beta-lactam resistance in Gram-negative bacteria, especially Escherichia coli, is a main clinical problem. It is often caused by the co-production of β-lactamases, particularly extended-spectrum b-lactamases (ESBLs) and AmpC enzymes. It may lead to a problem for diagnosis via recommended phenotypic tests by the Clinical and Laboratory Standards Institute (CLSI). Conversely, b-lactamase genes have several subfamilies; therefore, using designed primers with high ability could be valuable to detect all of them.Objectives: This investigation focused on evaluating the prevalence of extended-spectrum b-lactamases using disk diffusion method and confirmatory test (Combined Disk), as well as PCR for complete detection of these genes (SHV- and AmpC (CITM, FOX) - type b-lactamase genes).Materials and Methods: 500 clinical samples were collected from different hospitals of Tehran, Iran and 200 E. coli isolates were detected by standard biochemical tests such as IMVIC. These specimens were isolated from urine, stool, blood, and wound. Subsequently, the isolates were screened by disk diffusion method and combined disk assay for b-lactamase production. Resistant isolates were evaluated by PCR for molecular assessing.Results: In disk agar diffusion test, 128 (64%) E. coli isolates resistant to ceftazidime (55.5%) and cefotaxime (64%) were selected and followed by combined disk (ceftazidime, cefotaxime, and clavulanic acid) assay. In combined disk assay, among 128 screened isolates, 115 (89.8%) and 13 (10.2%) isolates were detected as ESBLs and AmpC producers, respectively. PCR performed on all 128 selected isolates via phenotypic tests and the results showed that among 115 and 13 isolates, 7 (6.1%) and 13 (100%) cases contained blaSHV and blaCITM, respectively. Fox gene was not detected in any sample.Conclusions: Our findings indicate that the prevalence of ESBLs in Tehran is rising. According to CLSI, isolates showing negative confirmatory test are potentially considered as producers of AmpC (the result of CITM PCR was 100% positive). On the other hand, co-production of ESBLs and AmpC may lead to ESBLs false negative. Thus, development of diagnosis methods for complete detection of b-lactamase enzymes is important for resistance control and the treatment with high achievement.