Background:  Pseudomonas aeruginosa (P. aeruginosa) is a globally recognized cause of healthcare-associated infections (HAIs), the recent increase of the MDR and XDR P.  aeruginosa strains encouraged the use polymyxins as a treatment option, and thus the emergence of colistin-resistant strain is an alarming problem. Objectives: This study aimed to trace the emergence of colistin-resistance in P.  aeruginosa strains associated with HAIs in Sohag University Hospitals, to identify the genetic basis of colistin-resistance in these isolates. Methods: P. aeruginosa strains were isolated and identified phenotypically and genotypically, antibiotic susceptibility of the isolates was tested by disc-diffusion method. The MIC of colistin was measured by E test in colistin resistant isolates. Conventional PCR was used to detect plasmid genes responsible for colistin resistance among the isolates. Results: Seventy-six(76%) of P. aeruginosa isolates were resistant to colistin, the highest percentage of colistin resistant strains were isolated from patients admitted to General Surgery Department that was (50%), no colistin resistant strains were isolated from patients admitted to Vascular Surgery Department. Colistin-resistant isolates exhibited the highest resistance to polymyxin B, norfloxacin, ofloxacin and gatifloxacin by a percentage of (100%). mcr-1gene was detected in (44.4%) of colistin-resistant isolates and mcr-2 gene in (16.6%). Sensitivity of E-test in comparison with PCR was (100%) and specificity was (86.36%). Conclusion: The emergence of colistin resistance in P. aeruginosa in our health care setting is an alarming issue that needs strict adherence to the infection control guidelines specially plasmid mediated resistance as it usually associated with MDR and XDR patterns.