Tuberculosis (TB) has killed millions of people throughout history and still isone of the leading causes of death. Since the early 1990s, ancient DNA(aDNA) research has made considerable contributions to the study of thisinfectious disease in the past. While early studies used polymerase chainreactions (PCRs) solely to identify the TB-causing organisms, namely theMycobacterium tuberculosis complex (MTBC), later approaches extended thefocus to assign the actual disease-causing species or strains of the MTBCbut were either directed at single or few individuals or only provided few data. This research project has screened a large set of European skeletaland dental samples from individuals of the 1st–19th centuries AD for IS6110,an insertion sequence believed to be specific to the MTBC, and has identifieda number of individuals that may indeed have suffered from TB. Reports ofIS6110-like elements in other mycobacteria, however, challenge thesuitability of IS6110 for detecting MTBC. Two sequences similar but notidentical to IS6110 were revealed from several of the samples analysed,supporting the proposal that IS6110 should not serve as the sole target foridentifying MTBC from archaeological material. It cannot be establishedwhere these sequences derive from, but application of a MycobacteriumspecificPCR and targeting of genomic regions of the MTBC that containsingle nucleotide polymorphism (SNPs) indicate that at least some of thesamples contain a range of unknown, most likely environmental, bacterialand/or mycobacterial species. Yet, screening for IS6110 together with thedetection of large sequence polymorphisms (LSPs) and SNPs in othergenomic regions has identified eight individuals to unambiguously containMycobacterium tuberculosis aDNA. Apart from one individual which wasrecovered from Northern France, these skeletons derived from Britisharchaeological excavation sites. The SNP and LSP results enabled theallocation of infecting MTBC strains into various classification systemsreported in clinical literature and revealed that M. tuberculosis strains variedthroughout different time periods, thereby mainly confirming evolutionarypathways suggested in previous studies. Additionally, it was found thatdistinct strains co-existed temporally, and maybe even spatially, in Britainand that at least one individual harboured two different MTBC strains,suggesting a mixed infection. Application of next generation sequencingenabled one of the 19th century strains from Britain to be characterised ineven more detail, revealing closest similarity to a M. tuberculosis strainisolated at the beginning of the 20th century in North America.