Extended-spectrum B-Lactamase (ESBL) producing organisms are among the fastest growing problems in our hospitals. The aim of this work was to study the genetic characterization of extended spectrum b-lactamase producing E.coli by detection of TEM-1,SHV-1,INT-1,DELTA ,CTXM-1 genes then to analyze the relationship between these isolates using Pulsed Field Gel Electrophoresis (PFGE) followed by bionumerical analysis to reveal if there is an outbreak of a specific strain. All strains showed high resistance pattern to ampicillin, gentamicin, tetracycline, trimethoprim, sulfonamides, quinolones, and third generation chephalosporins. All samples were positive for TEM-1 gene , SHV-1 gene ,80% of samples were positive for DELTA gene 60% of samples were positive for INT-1 gene ,All samples were negative for CTXM-1 gene by PCR .The selected isolates were subtyped by pulsed-field gel electrophoresis (PFGE) in accordance with the standardized E. coli protocol using (Xba I) restriction enzyme. No clonal relationship was found among the ESBL producing E.coli for most of the samples .In conclusion, ESBL producing E.coli isolates were found to be endemic in our hospitals with the threat of intra-hospital dissemination , control measures are Highly indicated.