Background: The growing incidence of infections caused by Colistin resistant gram-negative bacteria is seen as a serious public health concern. The CHROMID Colistin R agar is the first selective chromogenic agar to be validated for the screening of Colistin resistant microorganisms in clinical samples. Objective: The aim of the study was to assess the diagnostic performance of CHROMID Colistin R agar in detection of Colistin resistant gram-negative bacteria based on the different color of colonies. Methodology: This study was carried out between January 2020 and January 2021. Clinical samples were isolated from patients admitted to Mansoura university hospitals with different signs and symptoms of infection. The colony morphology, Gram stain, biochemical reactions, and Vitek-2 System were used to identify isolated colonies. Antibiotic susceptibility testing was performed by Vitek-2 System. Colistin resistant bacteria were further sub-cultured on CHROMID Colistin R Agar. Results: Out of 4800 collected samples, bacterial growth was detected in 15%. The colistin-resistant strains were collected from 51 patients. The most frequently isolated organisms were Acinetobacter baumanii (29.4%) followed by Pseudomonas (21.6%), Klebsiella pneumoniae (21.6%), and Escherichia coli (19.6%). There was a very good agreement between the two techniques in the detection of Colistin resistant Gram-negative bacteria (Kappa= 0.921, P-Value = 0.001). CHROMID Colistin R Agar had an overall sensitivity of 92.2%, 100% specificity, 100% PPV, and 99.9 % NPV. Conclusion: We concluded that CHROMID Colistin R agar is a reliable culture medium that can be used effectively for rapid screening of Colistin resistant Gram-negative bacteria.