The aim of this study was to determine the prevalence of ESBL-producing Escherichia coli and Klebsiella pneumoniae as well as genes encoding ESBLs. During this study, 1465 different clinical samples were tested in which 1255 (85.66%) samples showed growth of bacteria. Out of 1255 clinical samples, 1157 (92.19%) different strains of bacteria were isolated. Escherichia coli was the most prevalent 25.58% followed by Klebsiella pneumoniae (13.65%). Out of 296 E. coli isolates and 158 K. pneumoniae isolates, 247 (83.44%) and 128 (81.01%) isolates were ESBL producers, respectively. For testing with getotyping of isolates by Multiplex PCR detection using TEM, SHV, CTX-M genes, among the isolates harbouring single ESBL gene (61.53%), blaSHV, blaTEM and blaCTX-M were present in 42.30%, 19.23% and 11.53% strains of E. coli and K. pneumoniae, respectively. The 22 strains of E. coli and 16 strains of K. Pneumoniae had a single ESBL gene, although TEM and SHV types of ESBL were frequently found in E. coli (12/6) and K. pneumoniae (10/4), respectively. Two or more genes for ESBL were present in 14 (26.92%) of the 52 ESBL typeable isolates, blaTEM + blaSHV being the most common combination (9.61%), followed by blaTEM + blaCTX-M and bla SHV + blaCTX-M (5.76%). One strains of both E. coli and K. pneumoniae harbour 3 genes for ESBL. The majority of strains harboured two or more ESBL genes and the most common phenotypes were TEM, SHV and CTX-M. Identification of the genes is necessary for the surveillance of their transmission in hospitals.