Summary: Three medieval bone samples with osteological evidence of tuberculosis infection were analysed for the presence of DNA sequences from Mycobacterium tuberculosis using a series of PCRs. In each case amplification of IS6110 and part of the β-subunit of RNA polymerase identified infection with a bacterium belonging to the M. tuberculosis complex. Amplification of the mtp40 genome fragment and the presence of a guanine residue at position 285 in the oxyR pseudogene, demonstrated the infecting strain to be similar to present day M. tuberculosis isolates rather than to Mycobacterium bovis. Spoligotyping, based on amplification of the direct repeat (DR) region of the mycobacterial genome, provided further evidence of similarity to M. tuberculosis and indicated a close relationship between isolates associated with two separate medieval burials. The study demonstrates the feasibility of amplifying multiple M. tuberculosis loci in ancient human remains and suggests important applications in the study of the palaeoepidemiology and virulence of tuberculosis in past populations.