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Accurate serotyping is essential for effective infection control. Pseudomonas aeruginosa serogroup G is one of the most common serogroups found in water. Conventional serotyping methods are not standardized and have several shortcomings. Therefore, a robust method for rapidly identifying P. aeruginosa serotypes is required. This study established a real-time PCR method for identifying P. aeruginosa serogroup G strains using novel target gene primers based on comparative genomic analysis. A total of 343 genome sequences, including 16 P. aeruginosa serogroups and 67 other species, were analyzed. Target genes identified were amplified using real-time PCR for detecting P. aeruginosa serogroup G strains. Eight serogroup G genes, PA59_01276, PA59_01887, PA59_01888, PA59_01891, PA59_01894, PA59_04268, PA59_01892, and PA59_01896, were analyzed to determine specific targets. A real-time fluorescence quantitative PCR method, based on the novel target PA59_01276, was established to detect and identify serogroup G strains. The specificity of this method was confirmed using P. aeruginosa serogroups and non-P. aeruginosa species. The sensitivity of this real-time PCR method was 4 × 102 CFU/mL, and it could differentiate and detect P. aeruginosa serogroup G in the range of 4.0 × 103–4.0 × 108 CFU/mL in artificially contaminated drinking water samples without enrichment. The sensitivity of these detection limits was higher by 1–3 folds compared to that of the previously reported PCR methods. In addition, the G serum group was accurately detected using this real-time PCR method without interference by high concentrations of artificially contaminated serum groups F and D. These results indicate that this method has high sensitivity and accuracy and is promising for identifying and rapidly detecting P. aeruginosa serogroup G in water samples. Moreover, this research will contribute to the development of effective vaccines and therapies for infections caused by multidrug-resistant P. aeruginosa.