Background: Colistin has become a critical last-resort option for treating severe infections that remain unresponsive to other antibiotics, especially carbapenems. The rise in colistin resistance, along with the discovery of mobile colistin resistance (mcr) genes, significantly complicates the management of these infections. Determining colistin susceptibility can be challenging due to the drug's unique chemical properties and the various resistance mechanisms employed by bacteria. Aim: We aimed to assess the prevalence of colistin resistance among multidrug-resistant (MDR) Gram-negative bacilli (GNB) isolated from different clinical samples at Ain Shams University hospitals and for the molecular detection of different plasmid genes mediating such resistance (mcr-1 to mcr-10). Methods: We used the Colistin Broth Disk Elution Method (CBDE) to assess colistin susceptibility. Resistant strains were further subjected to a conventional polymerase chain reaction (PCR) assay to identify the genes mediating such resistance. Results: In total, we identified 12 isolates resistant to colistin by the CBDE test, with Klebsiella pneumoniae being the most resistant bacterial species. These resistant isolates were examined using conventional PCR which revealed the presence of multiple mcr genes, with mcr-2 being the most commonly detected. Conclusion: Our study highlights the concerning prevalence of colistin resistance among MDR GNB at Ain Shams University hospitals. The detection of multiple mcr genes in these isolates underscores the importance of continuous surveillance and molecular characterization of colistin resistance mechanisms.