Introduction One of the reasons for drug resistance in Escherichiacoli isolates is the production of broad-spectrum beta-lactamases. The widespread use of antibiotics in the agricultural and dairy industry has led to raisingin antibiotic resistance. Therefore, the aim of this study was to investigate the phenotypic and molecular ESBLs production among E. coli isolated from food. Materials and Methods This descriptive cross-sectional study was carried out by 93 E. coli isolated from food and poultry in Hamadan in 2017. Then, the microbial susceptibility of the beta-lactamase producing isolates was determined using the (Combined teat CT) method and according to CLSI (2015) guidelines. Also, molecular identification of genes producing ESBLs (blaSHV, blaCTX-1, and blaTEM-1) was performed by PCR method. Results Evaluation of microbial susceptibility showed that the highest antibiotic resistance was observed for nalidixic acid (%88. 4), ampicillin (%76. 8), tetracycline (%82. 8), and sulfomethoxazole (%67). Resistance to ceftazidime, Cefoxitin, aztreonam, cefotaxime was not observed in this study. The genotypic study by PCR method showed that the frequency of blaSHV, blaCTX-1, and blaTEM-1genes in E. coli isolated from food (%5. 37), (%19. 35) and (%29. 03), respectively. Conclusion E. coli isolated from food showed high resistance to antibiotics such as ampicillin, sulfomethoxazole, and tetracycline. On the other hand, the PCR method is more sensitive than the culture method.