Nontuberculous mycobacteria (NTM) frequently detected in drinking water (DW) include species associated with human infections, as well as species rarely linked to disease. Methods for improved the recovery of NTM DNA and high-throughput identification of NTM are needed for risk assessment of NTM infection through DW exposure. In this study, different methods of recovering bacterial DNA from DW were compared, revealing that a phenol-chloroform DNA extraction method yielded two to four times as much total DNA and eight times as much NTM DNA as two commercial DNA extraction kits. This method, combined with high-throughput, single-molecule real-time sequencing of NTMrpoBgenes, allowed the identification of NTM to the species, subspecies, and (in some cases) strain levels. This approach was applied to DW samples collected from 15 households serviced by a chloraminated distribution system, with homes located in areas representing short (<24 h) and long (>24 h) distribution system residence times. Multivariate statistical analysis revealed that greater water age (i.e., combined distribution system residence time and home plumbing stagnation time) was associated with a greater relative abundance ofMycobacterium aviumsubsp.avium, one of the most prevalent NTM causing infections in humans. DW from homes closer to the treatment plant (with a shorter water age) contained more diverse NTM species, includingMycobacterium abscessusandMycobacterium chelonaeOverall, our approach allows NTM identification to the species and subspecies levels and can be used in future studies to assess the risk of waterborne infection by providing insight into the similarity between environmental and infection-associated NTM.IMPORTANCEAn extraction method for improved recovery of DNA from nontuberculous mycobacteria (NTM), combined with single-molecule real-time sequencing (PacBio) of NTMrpoBgenes, was used for high-throughput characterization of NTM species and in some cases strains in drinking water (DW). The extraction procedure recovered, on average, eight times as much NTM DNA and three times as much total DNA from DW as two widely used commercial DNA extraction kits. The combined DNA extraction and sequencing approach allowed high-throughput screening of DW samples to identify NTM, revealing that the relative abundance ofMycobacterium aviumsubsp.aviumincreased with water age. Furthermore, the two-step barcoding approach developed as part of the PacBio sequencing method makes this procedure highly adaptable, allowing it to be used for other target genes and species. Copyright © 2018 Haig et al.