Infrequent restriction site amplification (IRS-PCR) is a method of amplifying DNA sequences, which flank an infrequent restriction site, and produces a strain-specific electrophoretic pattern. We studied the use of IRS-PCR to characterize Mycobacterium tuberculosis and non-tuberculous mycobactria (NTM). One-hundred and sixteen M. tuberculosis and nine NTM isolated at Hanyang University Hospital in Seoul, Korea were used in this study. IRS-PCR using AH1 and PX-G primers produced unique patterns for reference strains, M. tuberculosis H37Rv, M. bovis BCG, M. kansasii, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulae, M. fortuitum, and M. chelonae, respectively. Reference strains M. tuberculosis H37Rv, M. bovis, M. africanum, and all isolates of M. tuberculosis showed similar IRS-PCR patterns. The IRS-PCR patterns generated with multiple isolates of M. tuberculosis from the same patients were essentially identical. IRS-PCR revealed the greatest difference between electrophoretic DNA patterns from M. avium, M. intracellulae, and M. fortuitum that differed from each other and from the reference strains. We concluded that IRS-PCR is a useful tool for strain typing of NTM, but not for M. tuberculosis.