Escherichia coli (E. coli) plays a central role as an indicator for fecal contamination to predict the possible presence of microbial pathogens in drinking water. Current detection methods for E. coli are based on time-consuming culture-based techniques. There is a strong need for methods to detect fecal contamination rapidly in distributed drinking water to prevent outbreaks of waterborne disease and support water utilities to efficiently manage their operations like actions to repair or maintain distribution pipes, to minimize impact on consumers. This study describes the validation and application of a qualitative real time reverse transcription PCR (RT-PCR) method targeting 16S ribosomal RNA (rRNA) for rapid detection of E. coli in distributed drinking water. The RT-PCR assay targets 16S rRNA, a highly abundant RNA in viable cells, enabling robust detection at the required sensitivity of 1 CFU/100 ml. The validation was performed by comparing the RT-PCR method with the culture-based chromogenic reference method (CCA) using the protocol and criteria described in ISO 16,140–2:2016. The validation demonstrated that this RT-PCR method can be used to specifically detect E. coli in a broad range of drinking water samples with at least the same limit of detection as the culture method (Relative Limit Of Detection = 0.75, range 0.43–1.43). The inclusivity study showed that the RT-PCR method was able to detect a broad range of E. coli strains derived from different sources and geographic areas, including pathogenic serotype O157 strains that are not detected with the culture method. The exclusivity study determined that other bacterial genera are not detected with this RT-PCR. However, Escherichia fergusonii was detected and, based on “in silico” analysis, it is expected that also E. albertii and E. marmotae and Shigella species will be detectable using this RT-PCR. An interlaboratory study confirmed that the RT-PCR and culture method have comparable sensitivities when tested by different participants at different laboratories. The application of RT-PCR to confirm the hygienic quality of distributed drinking water after actions to repair or maintain distribution pipes was compared with the culture method on 8076 routine samples, analyzed by the drinking water laboratories in the Netherlands. This comparison study showed a 96.4 % agreement between RT-PCR and culture. In 3.3 % of the samples E. coli was detected with RT-PCR and not with the culture method and in 0.1 % of the samples E. coli was only detected by culture confirming either a higher sensitivity for RT-PCR or the detection of RNA from uncultivable cells. Finally, the application of RT-PCR was highlighted during a contamination event in Belgium where we demonstrate the potency of RT-PCR as a tool to rapidly monitor the spread of microbial contamination and to monitor the effect of measures to remove the contamination This is the first fully validated rapid nucleic based method for detection of E. coli in distributed drinking water. These results demonstrate that this RT-PCR method can be used as a rapid alternative to the culture method to monitor E. coli in distributed drinking water. However, it should be emphasized that nucleic acid based detection methods rely on highly different detection principles (detection of captured nucleic acids present in a sample) than culture base methods (presence of cells cultivable on a selective medium) resulting in occasional different analysis results. Varying treatment and disinfection steps (UV, chlorine, monochloramine, Ozone) or environmental factors (decay) can influence the results and cause differences between RT-PCR and culture methods.@en