Nucleic acid sequence capture extraction was coupled with LightCycler PCR amplification and product detection using real-time fluorescence for rapid, definitive detection of Mycobacterium bovis in lymph node specimens from 38 cattle with bovine tuberculosis lesions. PCR amplification of sequence-captured DNA using both a conventional heating block thermocycler and a LightCycler thermocycler was compared with culture and histopathological analyses. Conventional PCR enabled detection of 26 of 28 culture-positive specimens (93%) in approximately 9 h, and the LightCycler PCR detected 20 of 28 culture-positive specimens (71%) in only 30 min. Specific confirmation of Mycobacterium tuberculosis complex DNA was achieved by LightCycler PCR amplification using Syb Green 1 and an M. tuberculosis complex-specific Cy5-labeled fluorescence resonance energy transfer probe. The system described here enabled rapid and specific laboratory confirmation of bovine tuberculosis, and this is the first report of the detection of M. bovis in tissues using LightCycler PCR. The fluorescence technology used in the study has potential to allow development of a high-throughput molecular diagnostic test for bovine tuberculosis.