Colistin is considered as the last resort for treatment of bacterial infections caused by carbapenem-resistant Gram-negative bacilli (GNB). Colistin resistance can increase due to the spread of plasmid-borne mcr-1 gene. This study aimed to determine colistin susceptibility and to detect the presence of mcr-1 gene in the clinical isolates of GNB recovered from different clinical samples collected from Ain Shams University Hospital, Cairo, Egypt. About thirty-five GNB isolates were recovered from the different clinical samples that were collected during the period from February-April, 2019. These isolates were subjected to antibiotic susceptibility testing using disc diffusion assay, and colistin susceptibility through the E-test. In addition, conventional Polymerase chain reaction (PCR) was carried out for detection of mcr-1 gene among the colistin GNB resistant isolates. Most of the GNB isolates (60 %) were recovered from blood samples. Klebsiella pneumoniae (K. pneumoniae) was the most common isolated bacterium; that was represented by 24 isolates (68%). Out of the 35 GNB, only five isolates (14.3 %) were resistant to colistin by E-test, with MIC >256 𝜇g/ ml. The mcr-1 gene was detected only in one Pseudomonas aeruginosa (P. aeruginosa) isolate. This study concluded the low frequency of mcr-1 gene among the current GNB isolates. However, a large scale study is recommended to detect colistin resistance among GNB.