ABSTRACT The present investigation details an assessment of genetic relationship of E. coli isolates collected from two different environmental sources viz. sewage water and soiled bedding materials of laboratory rodents. Five sewage water samples were collected from the industrial area of Lucknow city and 5 samples of soiled bedding materials of laboratory animals were collected from Animal facility at CSIR-IITR, Lucknow. For this study Random amplified polymorphic DNA markers (RAPD) was chosen as the molecular fingerprinting method. In this study, 10 RAPD primers were used to evaluate the genetic similarity of E. coli. isolates. The RAPD-PCR fingerprints were analyzed and data were scored as 1, 0 matrix. The generated data were fed on Popgene software for calculating genetic diversity and creating dendrogram. The genetic similarity of 85% was recorded from soiled bedding materials and only 71% in sewage water samples in E.coli samples. The dendrogram based generation of clustering of E. coli isolates show two major clusters. Within major cluster sub-cluster were also observed which indicating diversity within isolates of E. coli. The RAPD-PCR based fingerprinting provided a rapid means of discriminating E. coli isolates and considered a relevant tool for molecular typing.