This study was conducted to assess the prevalence of resistance genotypes of extended-spectrum and AmpC β-lactamase producing Escherichia coli and Klebsiella pneumoniae. Out of 632 samples, suspected for E. coli and K. pneumoniae, collected from different units of Pakistan Institute of Medical Sciences, Islamabad, Pakistan, the number of positive samples for E. coli and K. pneumoniae were 593 (93.8 %). Out of these 593 isolates, 61.6% (n=365) were identified as E. coli and 38.4% (n=228) were K. pneumoniae. Common age group for sample isolation was 13-25 years for both E. coli and K. pneumoniae, from which 29.9% of E. coli (n=109) and 27.6% of K. pneumoniae (n=63) were isolated. However, none of the age groups achieved statistical significance. Higher percentage of E. coli was isolated from females as compared to males, while the ratio of K. pneumoniae was higher in male patients (p=0.012). Most of the isolates were recovered from specimens collected from outdoor patients and were mainly from urine samples. ESBL production was detected in 46.20% (n = 274) of the 593 isolates by phenotypic method. Out of total 365 E. coli strains, 49.3% (n = 180) were found to be ESBL producers and 41.2% (n =94) of total 228 K. pneumoniae isolates, were ESBL producers. Statistical analysis indicated that age groups have significant association with the presence of ESBLs (p= 0.007) in E. coli isolates. No significant association was observed in ESBL producing K. pneumoniae with age, gender, sample source or origin. AmpC β-lactamase production was detected in 25.8% (n = 94) of the total 365 E. coli and 20.6% (n =47) of total 228 K. pneumoniae isolates. There was significant association between males (p=0.018) and samples collected from surgical ward (p=0.01) with AmpC positive status in E. coli isolates. No significant association (p=0.88) was found in AmpC producing K. pneumoniae and gender. However, like AmpC producing E. coli, isolation from surgical wards had a statistically significant association with AmpC positive K. pneumoniae (p=0.001). Out of these 593 isolates, 200 samples of the phenotypically confirmed ESBLs or AmpC producers, E. coli and K. pneumoniae, were processed for antibiotic susceptibility analysis and detection of the selected genes. Out of 200, 131 were E. coli and 69 were K. pneumoniae. The highest resistance (90.1%) was observed against sulphamethoxazole, followed by tetracycline (88.5%) and ciprofloxacin (80%) among E. coli isolates. In case of β-lactam antibiotics, high resistance (87.8%) was observed against cefotaxime and amoxicillin/clavulanic acid, followed by cefepime (81.7%) and aztreonam (79.4%). Out of the total 131 E. coli isolates, 100 (76.3%) were found resistant to ceftazidime having an MIC >32μg/ml. Highest resistance was observed in case of amoxicillin/clavulanic acid, in which 117 isolates (89.3%) were resistant, followed by cefotaxime (116, 89.3%). About 45 (34.3%) isolates of E. coli showed resistance to cefoxitin with a maximum range of 256 μg/ml. PCR amplification revealed that CTX-M-1 was the most frequently (77 isolates, 58.7%) detected ESBL gene group, followed by TEM (25 isolates, 19%) and SHV (19 isolates, 14.5%). CTX-M-9 group was observed in only 4 bacterial isolates. Among AmpC β- lactamases, MOX gene was detected in 19 (14.5%) E. coli isolates, CIT in 17 (13%), CMY gene in 7 (5%), EBC gene in 5 (4%), and 2 isolates showed FOX AmpC β-lactamases. A total of 26 different patterns of genes were detected in 112 E. coli isolates, while no candidate gene was found in 19 E. coli isolates. Among K. pneumoniae, higher resistance was observed in case of tetracycline (98.6%), amoxicillin/clavulanic acid (97.1%) and sulphamethoxazole (95.7%). In case of β-lactam antibiotics, cefoxitin was the most successful antibiotic showing resistance to 20 (29%) isolates, followed by ceftazidime and cefepime (69.6%) and aztreonam (75.4%). MIC results revealed that fifty isolates (72.5%) were found resistant to ceftazidime with a maximum range of 512 μg/ml, while 19 (27.5%) were found susceptible. Fifty six (81%) isolates were resistant to cefotaxime and 61 (88.4%) to amoxicillin/clavulanic acid. Cefoxitin was the most successful antibiotic, effective against 47 (68.1%) of the total 69 K. pneumoniae isolates tested CTX-M-1 type ESBLs were detected in 43 (62.3%) isolates, SHV in 9 (13%), TEM in 8 (11.6%) and CTX-M-9 in 2 isolates (3%). Six (9%) isolates showed CIT type AmpC genes while 4 (6%) had CMY, 3 (4%) each FOX and MOX and 2 (3%) had EBC type genes. Eighty genes showed amplification in 69 K. pneumoniae isolates. A total of 18 different patterns of genes were detected in K. pneumoniae in a total of 58 isolates, while in 11 isolates, no gene was detected. Our study showed that both class A and class C β- lactamases contributed to cephalosporin resistance in E. coli and K. pneumoniae, thereby limiting therapeutic options. Co-expression of these enzymes may further hinder the identification of ESBLs, which is a critical step for designing a successful treatment for multidrug-resistant E. coli and K. pneumoniae. Sequence analyses revealed 99-100% homology with already reported ESBL genes from around the world. However, mutations in CIT gene were found which indicates possible amino acid substitutions in more than one position