We investigated the presence of nontuberculous mycobacteria (NTM) in three Mexican aquatic systems to evaluate the prevalence with the distribution of NTM species. Key physicochemical parameters of the water samples were determined to find correlations with the species' distributions. We used multilocus sequence analysis (MLSA) based on hsp65, rpoB, and 16S rRNA fragments to determine their taxonomic affiliations. NTM were recovered from water distribution systems and reclaimed water from the Mexico City Metropolitan Area (MCMA). The isolated species were associated with a temperature of 21°C and pH >7.7. The phylogenetic analysis showed that eight of the 14 different NTM strains were unambiguously classifiable: Mycobacterium peregrinum, M. nonchromogenicum (2), M. smegmatis (2), M. fortuitum, M. avium ssp. hominissuis, M. arupense, M. gordonae, and M. chitae. One strain was tentatively identified as M. mantenni/ scrofulaceum and another strain was related to M. porcinum/M. septicum. All NTM species identified in the water distribution system were also detected in the reclaimed water, but some species from the reclaimed water were not found in the water distribution systems. Two of the identified species found in the reclaimed water, M. avium and M. fortuitum, are considered important human opportunistic pathogens.