Colistin is considered the last option for treatment of infections caused by Multidrug Resistant (MDR) Gram negative pathogens. The mcr-1 gene could transfer the resistance to colistin between different species. Therefore, screening of this resistance mechanism will help greatly in the control of further spread of colistin resistance and enhancing the outcomes of patients. The current work aimed to study  the frequency of colistin resistance among MDR Gram negative pathogens isolated from clinical samples at Ain Shams University Hospitals and to explore if the mcr-1 gene was the responsible mechanism for this resistance. These pathogens were isolated from various samples including; blood, pleural fluid, sputum, urine, swabs from surgical and burn wounds, that were submitted from wards and intensive care units to the central microbiology laboratory at Ain Shams University Hospital, Cairo, Egypt, during the period from June to December 2019. Culture and bacterial identification were done by conventional microbiologic techniques. Eighty Gram negative bacterial pathogens were assayed for antimicrobial susceptibility by disc diffusion test. Sixty MDR Gram negative isolates were identified and further studied for colistin susceptibility by E- test, as well as real- time PCR to detect mcr-1 gene. Totally four isolates (6.7%) were phenotypically resistant to colistin. We found mcr-1 gene in three of the obtained isolates (5%); 1 Pseudomonas aeruginosa ( 5.3%) and 2 Acinetobacter baumannii (14.3%). In conclusion, although we detected a low prevalence of mcr-1 positive isolates from human clinical samples, continuous monitoring and implementation of infection control precautions are greatly required, to interfere with the further occurrence and transfer of bacterial species carrying the mcr-1 gene.