SRP342808 PRJNA773504 Distribution of bacterial pathogens within impaired watersheds Bacterial impairment of freshwater systems has been a commonly studied worldwide problem. However, studies on the relative distribution of bacterial pathogens within different impaired aquatic systems have been limited. Mostly, the impaired aquatic systems have been classified based on the presence of fecal indicator bacteria (FIB) and the identification of sources of fecal contamination through microbial source tracking. In the current study, we assessed the relative distribution of potential human pathogens along with the human fecal indicator's bacteria within three impaired watersheds. We used Illumina paired-end DNA sequencing of 16S rRNA gene amplicons from the three watersheds which consistently showed a high abundance of FIB for the past several years. We observed variation in the relative distribution of DNA sequences related to Legionellaceae, Enterobacteriaceae, and Bacteroidaceae families. We identified potential hotspots sites within these impaired water systems which showed a relatively high abundance of pathogen-related sequences. This study demonstrates the effectiveness of high-throughput DNA sequencing for the initial screening of waterborne pathogens and the identification of high-risk sites for preferential remediation efforts within impaired water systems. The frequent monitoring of specific pathogens that are in high abundance in a watershed can help in the accurate prediction and prevention of disease outbreaks.