Pipe-borne water is a major source of drinking water and contamination of such water source could lead to an epidemic. The aim of this present study is to isolate, identify and characterize microorganisms in pipe-borne water in different locations of Lagos metropolis. Six microorganisms were isolated from water samples collected at five locations. The isolates were subjected to morphological as well as biochemical tests using gram staining and Analytical Profile Index (API) tests respectively. Three primers (S gene, RAPD 1 and RAPD 2) were used for the PCR amplification of DNA samples extracted from the isolates and were identified as bacterial species: Bacillus polymyxa, Bacillus alvei, Bacillus mycoides, Pasteurella aerogenes, Pasteurella pneumotropica and Staphylococcus aureus.  Gel electrophoresis of the amplified DNA samples generated a total of 128 bands which were subjected to statistical analysis using the NTSYS software (version 2.02j). The results as shown in the UPGMA dendrograms revealed a similarity coefficient of 0.66 among B. polymyxa, B. alvei and B. mycoides as well as a high similarity coefficient of 0.72 between P. pneumotropica and S. aureus. The presence of spore-forming bacteria, B. mycoides B. polymyxa and B. alvei is considered a biohazard as they can survive for long periods while the presence of P. aerogenes, P. pneumotropica and S. aureus, known as opportunistic pathogens is equally hazardous and can be a potential cause of water borne disease. This present study, using RAPD-PCR technique has improved the identification and characterization of microorganisms isolated from pipe-borne water.