EXTENDED spectrum beta lactamases (ESBLs) of Escherichia coli and Klebsiella pneumoniae became a major problem in the whole world. This work aimed to study the prevalence of ESBLs genes (bla_TEM, bla_SHV and bla_CTX-M) in E. coli and K. pneumoniae clinical isolates recovered from Al Kasr Al-Ainy Hospital, Cairo, Egypt, during the period between 2016 and 2018. One hundred sixty eight clinical isolates were screened for ESBLs production by double disc synergy test (DDST) and combination disc test (CDT). ESBLs genes were detected by PCR using specific primers. Out of 168 isolates, 113 (67.26%) were phenotypically positive ESBLs. Genotypically, only 108 (95.58%) were confirmed as ESBLs producer (61 E. coli and 47 K. pneumoniae isolates). The percentage of bla_CTX-M, bla_SHV and bla_TEM were 91.7%, 82.4% and 78.7%, respectively. The co-existence of bla_TEM, bla_SHV and bla_CTX-M genes were found in 47.54 and 72.34% for E. coli and K. pneumoniae, respectively.