Aim: To develop quantitative PCR for culture-independent enumeration of enterotoxigenic Escherichia coli (ETEC) in sewage-impacted waters and aquatic weeds. Methods and Results: Two fluorescent probes (TaqMan and FRET) based on two different real-time PCR chemistries were designed in highly conserved region of LT1 gene encoding heat labile enterotoxin. Both the assays could detect 2 CFU ml-1 from serially diluted (two-fold and ten-fold) culture of reference strain (E. coli MTCC 723). FRET performed better in terms of C-T value and PCR efficiency than TaqMan. The presence of 106 CFU ml-1 of nonpathogenic E. coli reduced the detection limit two-fold with both the probes. However, the performance for two chemistries in various environmental samples was significantly (student's t-test, P < 0 center dot 05) different. Conclusion: It could be inferred from this study that real-time PCR chemistries (TaqMan and FRET) could detect very few copies of target DNA in pure cultures, but may give varied response in the presence of nonspecific DNA and natural inhibitors present in environmental sample matrices. Significance and Impact of the Study: The assays can be used for pre-emptive monitoring of aquatic weeds (a potential nonpoint source), surface and potable waters to prevent waterborne outbreaks caused by ETEC.