Frequency of extended-spectrum--lactamase-producing clinical isolates is increasing worldwide. This is a multi-center study which was aimed to check the frequency of third-generation cephalosporin resistance and distribution of the key genetic determinants of Extended-spectrum-β-lactamase-producing Clinical isolates in Pakistan.A total of 2372 samples were processed in three tertiary care hospitals and one diagnostic research center of Lahore, Pakistan during Aug-2014 to Sep-2017. Analytical profile index (API 20-E) was used for biochemical characterization of isolates. Antibiotic susceptibility testing (AST) and third generation cephalosporin resistant (3GC-R) isolates were subjected to: double disc synergism test (DDST), combination disc test (CDST) and epsilometric test (E-test) for confirmation of ESBL-production. PCR amplification of isolates with plasmid and genomic DNA was performed. Amplicon sequences were checked for gene-variants and statistical analyses were performed to check the significance of data.A total of 497/995 (50%) isolates including 65% ( = 321), spp. 25% ( = 124) and 5% ( = 24), 4% ( = 20) and spp. 2% ( = 8) were screened as third generation cephalosporin resistant (3GC-R). Urine 56% ( = 278) followed by pus 20% ( = 99) and wound swab 6% ( = 29) were frequent sources. Incidence of ESBL-producers detected by combination disc test was 79% ( = 392). PCR revealed (76%) gene followed by (52%), (28%) and (21%) were most prevalent among ESBL-producers detected by CDST. (65%), (78%) and (57%) genes were carried on plasmids. Amplicon sequencing revealed (75%), (49%) and (34%) and 21 ( = 28) isolates carried three genes in them.Prevalence of ESBL-producing isolates has increased 1.13 folds during study years. Isolates had high prevalence of ESBL-encoding gene and narrow spectrum and were also prevalent.