We evaluated the usefulness of three types of polymerase chain reaction (PCR) targeting 16S rRNA, protein antigen b and IS6110 in detecting Mycobacterium tuberculosis in preserved tuberculous lymph nodes. The detection limit of all PCR methods was 100 colony forming unit (CFU) of M. tuberculosis in tissue. The test samples included eight paraffin-embedded tuberculous lymph nodes containing microscopical epithelioid cell granuloma with caseous necrosis and Langhans giant cells. Although acid-fast stained organisms in lymph node tissue were not detected in any sample, all three types of PCR tests were positive in four of eight lymph nodes. Our results suggest that PCR is not only a rapid and sensitive diagnostic method for tuberculous lymphadenitis, but also clinically significant in retrospective study for detecting M. tuberculosis even in some preserved lymph node tissues without evidence of acid-fast stained organisms.