Background: Many extended-spectrum β-lactamases (ESBLs) variants are known among Gram-negative bacilli, and are classified into different structural families as TEM, SHV, CTX-M, and OXA. Objectives: To detect the distribution of blaTEM and blaCTX-M genes among the Gram-negative isolates collected from urine samples from patients admitted to ICUs of Ain Shams University Hospitals, Egypt. Methodology: Forty Gram-negative urinary isolates were enrolled, and subjected to microbiological identification and antimicrobial susceptibility testing. Phenotypic detection of ESBLs production was done. Detection of blaTEM and blaCTX-M genes was done by PCR. Results: Phenotypically, 8(20%) isolates were ESBL producers and 32(80%) were non ESBL producers. BlaTEM gene was found in 15 isolates(37.5%) and blaCTX-M gene was found in 8 isolates(20%), while both genes were detected among five isolates. Conclusion: Molecular methods should be used for definitive identification of ESBLs. BlaTEM gene was more common than blaCTX-M gene in urine specimens in our setting.