Background: Carbapenem is a beta-lactam antibiotic that works similarly to Penicillins and Cephalosporins by binding to Penicillin-binding sites and subsequently inhibiting cell wall synthesis. Due to their high potency and extraordinarily broad antibacterial action, Carbapenem antibiotics, which belong to the most recent generation of β-lactam antibiotics, are frequently used in clinics to treat bacterial infections. The current study aimed to shed light on Escherichia coli's resistance to β-lactam antibiotics, especially Carbapenem, and the potential link between Carbapenem resistance and the production of β-lactamase variants bla (NDM-1, NDM-2, NDM-3, NDM-4, NDM-5, GIM-1, VIM-1, SPM-1, and IMP-1) in Karbala province, Iraq. Materials and methods: Four hundred-eight isolates have been identified according to traditional methods (colonial morphology on MacConkey and Eosin methylene blue (EMB) agar, and biochemical reactions), and confirmed by the Vitek2® system. Carbapenem susceptibility was assayed by using the Vitek2® system. Isolates were subjected to multiplex PCR targeting β-lactamase variants genes. Results: Out of a total of 408 samples randomly collected from different clinical sources, 24 (19.51%) Escherichia coli isolates were diagnosed. Four (16.67%) of the Escherichia coli isolates tested positive for imipenem and meropenem resistance. PCR experiments indicated that only two (50%) isolates contained the blaNDM-1 gene and four (100%) carried the blaNDM-3 gene; no additional beta-lactamases were found.