Background: Colistin resistance in Gram-negative bacteria poses a significant threat to public health. This study investigated the prevalence of colistin resistance mediated by the PhoP gene in Gram-negative bacteria isolated from various water sources in the Ho Municipality, Ghana. Methods: Water samples were collected from different sources including boreholes, tap water, stored water, rainwater, streams, and wells. Gram-negative bacteria were isolated and identified using standard microbiological techniques and whole-genome sequencing. Colistin susceptibility was determined by broth microdilution. The presence of mcr genes was assessed by PCR, while PhoP was detected using nanopore sequencing. Results: Out of 132 water samples, 105 (79.55%) yielded Gram-negative bacterial growth. Phenotypic colistin resistance was observed in 52.1% of isolates, with 62.3% of resistant isolates carrying the PhoP gene. No mcr genes were detected. The prevalence of PhoP varied significantly among water sources, with direct tap water, stored tap water and well water showing the highest rates. Klebsiella spp., Proteus spp. and Pseudomonas aeruginosa were the most common PhoP-positive isolates. Conclusion: The high prevalence of PhoP-mediated colistin resistance in diverse water sources highlights the need for improved water treatment and sanitation practices. This study emphasizes the importance of environmental surveillance in understanding and controlling the spread of antibiotic resistance