Abstract Background: The incidence of infections caused by beta-lactam resistant organisms has increased in recent years due to the production of various enzymes. Extended-spectrum β-lactamases producing Enterobacteriaceae (ESBL-E) are emerging worldwide in hospitals and in the community. Aim of the work: The present study was done to determine the prevalence and molecular typing of extended-spectrum β-lactamases (ESBLs) in clinical isolates of Escherichia coli, Klebsilla pneumonae, Enterobacter cloacae, and Citrobacter freundii, isolated between January 2014 and August 2016 from the department of Oncology and Hematology, Zagazig University Hospitals. Patient & Method: Antimicrobial susceptibility testing was determined by Vitek2 & disk diffusion on Mueller Hinton agar for selection of the best antibiotic to be used in resistant cases. The characterization of ESBL genes were examined using PCR amplification and the clonal relatedness was investigated by ERIC-PCR. Results: During the study period, twenty-two (11%) isolates out of 420 specimen were found to produce ESBLs and distributed as follows: 12 isolates of E. coli (11%), 5 isolates of K.pneumonae (9.6%), 3 isolates of E. cloacae (9.37%) and 2 isolates of C. freundii (25%). ERIC-PCR was used to identify clonal relatedness, which proved that the isolates were genetically unrelated. The CTX-M-15 ESBLs were predominant representing (81.8%) of ESBL positive cases, followed by TEM-4 (18.2%) and SHV-12 (13.6%). A common band was observed in all extracts with a high molecular weight which is found to be CTX-M15 gene. The blaCTX-M-15 gene is transferred on a conjugative plasmid of high molecular weight (≈130 kb). Our results showed that all 22 strains (100%) were susceptible to imipenem, amikacin, colistin, fosfomycin and Cefepime/clavulanic Acid. Higher susceptibility levels were shown with Piperacillin/Tazobactam (90.9%) and Meropenam (81.8%). Conclusion: We conclude that there is high rate of multidrug resistant strains in our hospital with CTX-M-15 being the most common cause for Enterobacteriaceae ESBL resistance, followed by TEM-4 and SHV-12.