Aim: The increasing burden of resistance in Gram-negative bacteria (GNB) is becoming a major issue for hospital-acquired infections. Therefore, understanding the molecular mechanisms is important. Methodology: Resistance genes of phenotypically colistin-resistant GNB (n = 60) were determined using whole genome sequencing. Antimicrobial susceptibility patterns were detected by Vitek®2 & broth microdilution. Results: Of these phenotypically colistin-resistant isolates, 78% were also genetically resistant to colistin. Activation of efflux pumps, and point-mutations in pmrB, and MgrB genes conferred colistin resistance among GNB. Eight different strains of K. pneumoniae were identified and ST43 was the most prominent strain with capsular type-specific (cps) gene KL30. Discussion: These results, in combination with rapid diagnostic methods, will help us better advice appropriate antimicrobial regimens.