Background: A major problem in the treatment of the infectious diseases healthcare centers is extended-spectrum beta-lactamase (ESBL)-producing bacteria. Objectives: The aim of present study was to identify the antibiotic sensitivity pattern and prevalence of the blaCTX, blaTEM, and blaSHV genes in Escherichia coli and Klebsiella pneumoniae strains. Methods: In this study, E. coli and K. pneumoniae specimens were collected in Shushtar hospitals, Khuzestan (southwest Iran), from March to October 2015. Sensitivity antibiotic pattern performed by disc diffusion method. Double disc synergy test (DDST) done for identifying ESBLs isolates and PCR for blaTEM, blaSHV, and blaCTX-M genes. Results: One hundred E. coli and 30 K. pneumoniae isolates were collected from different specimens. The highest rates of antibiotic resistance related to cefotaxime and aztreonam in E. coli and K. pneumoniae. ESBL-harboring K. pneumoniae and E. coli were 13.5 and 28%, respectively. Overall, bla TEM was the most prevalent ESBL gene. Conclusions: In this study, the rate of antibiotic resistance was high, and due to the carrying of coding genes on mobile genetic elements and the ability of these elements to carry genes that create resistance to other antibiotic families, identification and isolation of these isolates are essential to find effective antibiotics and eliminate the infection.