Extended spectrum beta lactamases are rapidly evolving group of beta lactamase enzymes produced by enterobacteriaceae species. These extended spectrum beta lactamases (ESBL) have high resistance to the wide variety of antibiotics that are mostly used in hospitals where there has been increased concern over infections caused by E.coli. Totally 90 clinical isolates of E.coli were collected from local Government Hospital during Jan 2008 to April 2010. All suspected isolates were screened by disk diffusion synergy method and subsequently isolates showed the presence of ESBL were subjected to polymerase chain reaction (PCR) targeting TEM (temoniera) and CTX-M (cefotaximase) genes. Among the 80 isolates phonotypical 62 isolates confirmed presence of ESBL. Molecular analysis showed in those 62 isolates 37 showed presence of CTX-M gene and 24 showed presence of TEM gene, which was further restricted using restriction enzymes Hind III and Pst I for CTX-M and TEM gene respectively. From the results it was revealed that the high percentage of ESBL genes among the clinical isolates of E. coli.