Introduction: Gene amplification methods are increasingly used in diagnostic microbiology and the first applications for the detection of bacterial antibiotic resistance have been described. The aim of this study was to compare the disk-diffusion method and polymerase chain reaction for detection of antibiotic resistance Escherichia coli. Methods: During the period from June 2010 to September 2011, 250 stool samples were collected from children suffering from diarrhea in three hospitals in Tehran, Iran. Diarrheagenic E. coli isolation was carried out by standard procedures, and identification was achieved through biochemical and bacteriological methods. Disk diffusion methods and molecular analysis of antibiotic resistant genes (Tetracycline and ESBL resistance genes) by Multiplex PCR were compared. Results: According to number of specimens examined, 154 (26.37%) E. coli were isolated. The results of antibiotic resistance genes indicated that among all isolates that resistance to antimicrobial by disk diffusion tests harbors more than 70% their attribute resistance genes. Among antibiotic resistance genes, ampicillin with 88% was the highest rate resistance gene. Conclusions: The results showed that the disk diffusion method is a reliable method for the detection of E. coli antibiotic resistance isolates. Moreover, using application of disk diffusion method beside molecular analysis of antibiotic resistance genes enhance detection of the antibiotic resistance isolates.