Microbiological control of hemodialysis fluid is important for the prevention of hemodialysis-associated illness. Bacterial populations inhabiting a distribution system for hemodialysis water were studied over a 4 month period in five hospitals (one in Tehran, and the others at Alborz). All the samples from the four hospitals at Alborz had colony counts of ≥100 CFU/ml, which at different points of sampling were higher than the maximum recommended values. A total of 80 samples taken at different points in each hospital's hemodialysis distribution system were collected, and 229 planktonic bacteria isolated on R2A medium. No growth was detected by culturing the samples on Blood agar or Mueller-Hinton agar, according to routine procedures currently used in the five hospitals. A representative of isolates from each of 45 different morphotypes were identified using 16S RNA sequencing. A diverse bacterial community, containing predominantly gram-positive members of Kocuria, Arthrobacter and Staphylococcus and Mycobacterium, was detected. Bacteria from the genera Acinetobacter, Burkholderia, Halomonas, Herbaspirillum, Pseudomonas, and Sphingomonas were identified, which has been described in the build-up of biofilms. Some of the species reported here may represent a health risk to patients receiving hemodialysis treatment. In conclusion, it is recommended that standard protocols for evaluation of microbial contamination be used for regular monitoring and identification of culturable bacteria.