A 441-bp fragment of the 65-kD heat-shock protein (hsp65) was sequenced to gather taxonomic information about non-tuberculous mycobacteria (NTM) in two drinking water distribution systems: a chloramine-containing system in the United States and a system in Norway with no residual disinfectant in the drinking water supply. NTM are environmentally occurring members of Mycobacterium spp. that may act as opportunistic pathogens in humans. We aimed to characterize the NTM in the drinking water and water-main biofilms from the two systems to determine whether pathogenic species were present.