Pseudomonas aeruginosa has emerged as a major pathogen in nosocomial infections. Biofilm formation allows the microorganism to persist in hospital water systems for extended periods, which have been associated with nosocomial infections. The aim of this study was to evaluate the frequency of P. aeruginosa colonization of hospital tap waters by nested PCR assay. A total of 44 water samples were collected from 11 hospitals and analyzed for the presence of Pseudomonas spp. and P. aeruginosa by specific primer sets of 16S rRNA gene. Some physicochemical parameters and heterotrophic plate count (HPC) of samples for possible association with P. aeruginosa contamination were also determined. The nested PCR revealed 32% of the water samples being positive for P. aeruginosa. From the 11 hospitals surveyed, 82% (9 of 11) of the hospitals water systems were positive for P. aeruginosa. No correlation was seen between the presence of P. aeruginosa and HPC as well as physicochemical parameters. Identification of contaminated sources could be a key priority in waterborne nosocomial infections. PCR assay was used in the study provides simple, rapid, and reliable identification of P. aeruginosa in hospital water systems, which could eliminate the infections of P. aeruginosa through implementation of immediate control measures.