Identifying organisms that harbor extended spectrum beta lactamases (ESBLs) is a major challenge for a diagnostic clinical microbiology laboratory. The present study evaluated phenotypic characteristics, initial screening tests and established confirmatory phenotypic methods for detection of ESBLs Klebsiella isolates prevalent in Cairo university hospital. Standard disk diffusion, double disk synergy test (DDST), combined disk method, E test ESBL strip for detection of ESBL expression, as well as PCR analysis for detection of TEM1 and SHV1 genes were done. Results of screening of Klebsiella species using oxyimino-cephalosporin disk diffusion method showed 100% resistance (ESBL suspected) to cefpodoxime, 98% to cefotaxime, ceftriaxone and aztreonam, 96% to ceftazidime, and 92% to cefepime. However DDST method detected the presence of ESBL activity in 76% of isolates, and 98% by the combined disk diffusion test. The ESBL activity was detected by E-test method in 46 isolates (92 %). TEM1 gene was determined by PCR in (96%), and SHV1 was determined in (90%) of cases. The combined disk diffusion test is an easy reliable method which showed the highest incidence of ESBL detection in our study followed by E test. However the DDST method showed the lowest ESBL detection rate in comparison to the aforementioned confirmatory tests. The presence of TEM1 and SHV1 genes was confirmed in a high incidence of ESBL isolates .