Extended-spectrum beta-lactamase (ESBL) production is the most important cause of betalactam resistance in Gram-negative bacteria. Although it may also be found in other Gramnegative bacteria, ESBL is most commonly produced by Escherichia coli and Klebsiella pneumoniae strains. In this study, we aimed to investigate the distribution of β-lactamase genes in ESBL-producing E. coli and K. pneumoniae strains. One hundred and twenty isolates of E. coli and K. pneumoniae isolated from clinical samples were used in this study. The identification and the antibiotic susceptibility tests were performed by VITEK 2 system in accordance with the manufacturer’s instructions. ESBL production was determined accoring to Clinical and Laboratory Standards Institute guidelines. The DNA isolation was performed with a commercial kit following company recommendations. blaTEM, blaSHV and blaCTX-M genes were amplified by multiplex PCR with specific primers. Of the 120 isolates collected, 84 isolates were of E. coli and 36 isolates were of K. pneumoniae. blaTEM gene was the most prevalent type (85.8%) followed by blaCTX-M (83.3%) and blaSHV (24.2%). No blaSHV gene was detected in the E. coli strains. Among 120 ESBL-producing strains, 10.8% were susceptible to cefepime, 10.0% to ceftazidime, while 5.0% to ceftriaxone. In conclusion, blaTEM gene was the most frequently encountered ESBL of E. coli and K. pneumonia in our hospital. Further molecular surveillance and epidemiological studies of such resistant bacteria are recommended for monitoring and controlling the spread of ESBL producing strains.