Colistin-resistant Pseudomonas aeruginosa (CRPA), as a health care system threat, is increasing globally. This study aimed was to determine CRPA by High-Resolution Melting Curve (HRM) Analysis method. The HRM method was done on standard strains of P. aeruginosa and CRPA strains. Nine-fold serial dilutions of known genomic DNA (gDNA) and plasmid DNA (pDNA) concentrations, extracted from P. aeruginosa NCTC13715 and P. aeruginosa NCTC10728 were prepared and tested by melting curve analysis and HRM assay. Data analysis was performed using the Step-One Plus Software v2·3 and HRM Software v3·0.1. The results of the melt curve analysis and HRM showed a very similar melt peak for all positive controls with a melt temperature that ranged from 88·1°C to 88·6°C for the 16srRNA, 90·0°C to 90·05°C for the mcr-1 and 91·2°C to 91·7°C for the pmrA gene. Furthermore, the data indicated that the HRM approach has the sensitivity to detect 10  CFU/mL for the 16srRNA gene, 10  CFU/ml for the pmrA gene, and 10  CFU/mL for the mcr-1 gene. According to our findings, it was concluded that the HRM method could detect 10 to 10  CFU/mL of P. aeruginosa gDNA and pDNA. Therefore, CRPA strains can be identified with high sensitivity and specificity by HRM assay.