Most microbiological water quality regulations rely upon the detection of indicators of fecal pollution, such as coliform bacteria, or more specifically Escherichia coli. In order to further understand the source, fate, and implications for water quality regulation, environmental E. coli isolates should be assessed genetically to observe various levels of genotypic diversity. Multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) is a novel, simple and inexpensive polymerase chain reaction (PCR) based genotyping method which relies on the detection of different copy numbers inside each VNTR locus. In this study, we evaluated MLVA as a tool for the genotyping of E. coli strains of water samples collected from the Karaj River, Iran. Overall, high genetic diversity was observed among environmental E. coli isolates. We also proved the feasibility of MLVA as a complementary or even future replacement genotyping method for the average routine laboratory.