The emergence and spread of carbapenem resistance in Gram-negative bacilli such asKlebsiella pneumoniae,Escherichia coli,Acinetobacter baumannii, andPseudomonas aeruginosathrough the production of carbapenemases is a global phenomenon. It threatens patient care and leads to therapeutic impasses. This study aims to genotypically determine the prevalence of the most frequent carbapenemase genes among multidrug-resistantE. colistrains isolated from patients at a biomedical analysis laboratory. A total of fifty-three unduplicatedE. colistrains isolated from patient samples with a multidrug-resistant (MDR) profile were subjected to polymerase chain reaction (PCR) testing for carbapenem resistance genes. This study allowed us to identify fifteen strains carrying resistance genes among the fifty-threeE. colistrains. All fifteen strains produced the metallo-β-lactamase enzymes; this represents a rate of 28.30% of study strains. Among these strains, ten carried the NDM resistance gene, NDM and VIM genes were detected in three strains and VIM was identified in two strains ofE. coli. However, carbapenemases A (KPC and IMI), D (OXA-48), and IMP were not detected in the strains studied. Thus, NDM and VIM are the main carbapenemases detected in the strains in our study.