Opportunistic pathogens (OPs) pose significant health risks in poorly operated distribution systems (DSs) or outside the water utility’s jurisdiction in customers’ premise plumbing systems. Due to their complex ecological habitats and resistance to disinfectants, it has been difficult to understand and characterize occurrence and dissemination of these OPs (specifically Legionella pneumophila, Pseudomonas aeruginosa, and non-tuberculous mycobacteria [NTM]) in drinking water distribution systems (DWDSs). This research aimed to develop a practical sampling and analytical approach for water utilities to quantify OPs presence in DWDSs such that actionable data can be gathered for future risk determination and mitigation. Co-funding provided by AWWA Government Affairs Office. Published in 2023.