Aim: Nosocomial infections have become increasingly a major health concern in many hospitals. Gram-negative bacteria (GNB), including Acinetobacter baumannii, Pseudomonas aeruginosa and Legionella have emerged among the most problematic microorganisms in hospital settings, which can cause a variety of nosocomial infections, especially in susceptible individuals. Biofilm formation allows these waterborne agents to persist in hospital water systems for extended periods. Since the transmission is the initial step in disease occurrence, effective prevention of nosocomial infections requires a better knowledge about waterborne bacteria. The aim of this study was to investigate the frequency of presence of GNB in hospital water systems by a rapid and reliable assay. Materials and Methods: A total of 33 water samples were collected from 11 hospitals of Isfahan University of Medical Sciences, Iran and analyzed for the presence of GNB by a polymerase chain reaction (PCR) assay with the application of specific primer sets. Results: From the 11 hospitals surveyed, 91% (10 of 11) were positive for at least one of the types of GNB. GNB were detected in 58% (19 of 33) of water samples. 45% (15 of 33) of samples were positive for legionella. A. baumannii and P. aeruginosa were detected in 18% (6 of 33) of water samples. The mean concentration of heterotrophic bacteria was 36 CFU/ml. Conclusion: Detection of GNB in hospital water systems with a relatively high frequency revealed that hospital water may act as an important route for transmission of nosocomial infections. The results emphasize the importance of rapid microbiological monitoring and the implementation of strict control measures in hospital water systems.