Aims: Carbapenemase-producing bacteria make infections of the urinary tract (UTIs) challenging to cure with last-resort treatment like carbapenem. Carbapenemase-producing E. coli and K. pneumoniae implicated in UTI must be detected molecularly since their ability to spread broadly among patients is rising (Nomeh et al., 2022). Methodology: Ten non-repeated clinical isolates of Escherichia coli (Ecoli1, Ecoli2, Ecoli3, Ecoli4, and Ecoli5) and Klebsiella pneumoniae (Kp6, Kp7, Kp8, Kp9, Kp10) were selected from urinary tract infection patients at Institute of Kidney Disease Peshawar, Pakistan, based on their in vitro phenotypic carbapenem antibiotic resistance. These isolates were confirmed using standard routine microbiological techniques. PCR-specific primers screened E. coli and K. pneumoniae strains for Carbapenemase-producing genes. Result: Molecular Detection of Carbapenemase-producing Gene in UTI Patients with Uropathogenic Escherichia coli and Klebsiella pneumoniae. The higher proportion of Carbapenemase-producing genes in all the bacterial isolates in this study was blaKPC 15(100 %), followed by blaNDM 12.3(90.1 %), blaIMP 6(60.2 %) and blaVIM 3(30.6 %). The most common Carbapenemase gene in Escherichia coli 8 (80%) was blaKPC, followed by blaNDM 7 (70%) and blaOXA 45 (4.5%), which was the least common. Klebsiella pneumoniae had more blaNDM and blaKPC than blaOXA. Both had a percentage of 4.5 (40.9%). Conclusion: These results are consistent with the rapid spread of genes responsible for generating Carbapenemases in E. coli and Klebsiella pneumoniae that cause urinary tract infections. Despite the lack of blaVIM in K. pneumoniae, the pathogenic function of Carbapenemase-producing genes in UTI in this study should not be underestimated because of the potential they have to cause treatment failure and the subsequent persistence of UTI in patients.