Resistance to a wide variety of common antimicrobials is observed among clinical strains designed as extended spectrum β-lactmase (ESBL) producers. They produce enzymatic protein which inactivates efficiently oxyimino cephalosporin and constitutes a serious global health concern that has complicated treatment strategies. Many studies report high prevalence of ESBL producers among Gram negative bacilli. The aim of this work was to identify the presence of TEM, SHV and CTX-M families in these strains which were initially screened by phenotypic method. Gram negative bacilli resisting third or four generation cephalosporin were isolated during anti-biogram study. The presence of ESBL positivity was detected using the double disk synergy test. Minimal inhibitory concentrations (MICs) of ceftriazon for any strain were determined using E-test manufacturing protocol. Polymerase chain reaction (PCR) analysis for β-lactamase (bla) genes of TEM, SHV and CTX-M family was carried out using designed primers in 171 ESBL isolates producers. Among 259 Gram negative bacilli collected, 171 (66, 02%) exhibited ESBL producers' profile. Urine samples constitute major source of ESBL producers. The highest prevalence of ESBL was observed in Escherichia coli (75, 50%). Among ESBL isolates producers, gene prevalence of bla-CTX-M (65, 49%) was highest, followed by bla-TEM (25, 73%) and bla-SHV (18, 71%) in the present study. The frequency of ESBL producing strains among clinical isolates has been steadily increased. Continual drug resistance surveillance and molecular characteristics of ESBL isolates are necessary to guide the appropriate and judicious antibiotic use.