Background: Currently, a final-resort therapy for MDR Klebsiella pneumoniae and Escherichia coli is the antibiotic colistin (polymyxin E). Plasmid-mediated colistinresistance genes (mcr-9, mcr-8, mcr-5, mcr-3, mcr-2 and mcr-1) is one of the mechanisms by which these bacteria resist colistin. The aim of the study is to evaluate the presence of these genes in isolates of Klebsiella pneumoniae and Escherichia coli that are phenotypically colistin-resistant in a number of hospitals in Khartoum State. Materials and Methods: One hundred eighty-five bacterial strains (gram-negative rods) were identified from clinical specimens of male and female hospitalized patients. Various patients' urine, wounds, tissues, and blood samples were obtained. Isolates were identified using conventional methods of identification. Antimicrobial susceptibility testing for colistin was conducted using the disc diffusion method. Colistin-resistant K. pneumoniae and E. coli isolates were isolated and conventional PCR was employed to identify plasmid-mediated mcr-9, mcr-8, mcr-5, mcr-3, mcr-2 and mcr-1genes. The Chi-square test was administered, and p-values of 0.05 were deemed statistically significant. Results: The disc diffusion method revealed that around 35 (18%) of 185 isolates were resistant to colistin. 21 (60%) of the 35 colistin-resistant isolates were E. coli, and 14 (40%) were K. pneumoniae. All 35 colistin-resistant isolates and 13 colistin-susceptible isolates were examined for colistin-resistant genes. In 21 of 48 tested isolates, colistin-resistant genes were found. 14 (66%) of 21 isolates were resistant according to both the disc diffusion and PCR tests. There were determined to be mcr-3, mcr-2, and mcr-1 gene. mcr-1 accounted for 18 (85%), 5% mcr-1 combined with mcr-2 , 5% mcr-2 , and 5% mcr-2 in combination with mcr-3. Conclusion: The frequency of colistin-resistant E. coli and K. pneumoniae in Sudanese hospitals was found to be worrisome, thereby reducing the range of available treatment alternatives. The emergence of the colistin-resistant genes mcr-2 and mcr-1 in some Sudanese hospitals is caused by plasmids, and this is a problem for hospitals because horizontal transfer of this plasmid could cause the resistance to spread to many isolates there.