To investigate the occurrence and species diversity of mycobacteria in waters, surface water samples were collected monthly from the Han River and tap water samples at the terminal sites of the distribution system. Mycobacteria in each water sample were isolated by decontamination using cetylpyridinium chloride (CPC) and cultivation on Middlebrook 7H10 agar, and then identified by polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) and sequencing of the 65-kDa heat-shock protein gene (hsp65 gene). Mycobacteria were detected in 59% of the surface water samples and 26% of the tap water samples. Over half of the 158 isolates could not be identified by hsp65 PRA and gene sequencing, and several identification discrepancies were observed between the two methods. The most frequently isolated species was Mycobacterium gordonae in surface water and M. lentiflavum in tap water. M. avium complex (MAC), the most important pathogen among environmental mycobacteria, was detected in the surface water samples but not found in the tap water samples. The result demonstrated that water is an important environmental source of mycobacteria and the combined application of hsp65 PRA and sequencing was more reliable than hsp65 PRA alone to accurately identify mycobacteria present in water.