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Objectives: To evaluate extended spectrum betalactamase (ESBL) in E.coli and Klebsiella pneumoniae in bacterial cultures and its frequency at LRH. Study Design: Cross sectional analytical study. Setting: Lady Reading Hospital, Peshawar. Period: June 2013 to December 2013. Methodology: Total of 1037 bacterial isolates including 614 E.coli and 423 of Klebsiellapneumoniae were evaluated. All cases were subjected to double disc diffusion method for ESBL detection using amoxacilln-clavulanic acid and a third generation cephalosporin as all ESBLs are hydrolysed by clavulanic acid. The data were analysed using SPSS-16. Results: Out of 1037 cases five ninety two (55%) were males and four fourty five (45%) were females. Of these, E. Coli were 614 (59.2%) and K. Pneumoniae were four twenty three (40.8%). Of these 1037 isolates, four hundred & ninety five(47.7%) tested positive for ESBL enzyme. Frequency of ESBL positivity in E.coli isolates was 264 (43%) and in Klebsiellapneumoniae isolates was 231 (54.6%).Frequency of ESBL in pus was 34.3%(152/395),in urine, it was 31.8%(141/368), in blood it was 28.6%(127/233) and in sputum it was 5.1% (23/41). Unit wise frequency of ESBL was surgical & allied 24.6% (109/283), medical and allied 21.4%( 95/241), paediatrics 18.5% (82/203), obstetrics & gynaecology 23.2%( 103/178) and outpatients 12.1 %(54/132). No significant correlation between ESBL positivity, gender, unit or specimen was found. Conclusion: ESBL positive isolates of E.coli and K.pneumoniaeshould be properly detected in routine laboratory workflow to avoid unnecessary use of otherwise effective antibiotics.These results indicate that such organisms are highly prevalent in our Hospital and need immediate infection control measures to reduce their further spread. [ABSTRACT FROM AUTHOR]