Ancient pathogenomics is the field that studies past pathogens by recovering ancient DNA from archaeological remains. This discipline has been highly reliant on phylogenetic analyses, which provide information on: how past strains are related to their modern relatives; past diversity of the pathogen in question; and their geographical dispersal during past epidemics. However, in order to understand what genomic changes contributed to the dispersal, adaptation, and virulence evolution of pathogens, as well as to understand their past ecology, one needs to employ other analytical tools. In this thesis, I have employed and adapted the concepts of gene content, de novo assembly, and pangenomics to study the differences in virulence and functional potential of ancient bacterial strains. I have applied these approaches to Yersinia pestis, the bacterium responsible for plague, with a focus on its early genomic evolution in Eurasia between 5,000 to 3,000 years ago. I also demonstrate these concepts by applying these workflows to the first recovered ancient Streptococcus mutans genome from South Africa. This pathobiont is strongly associated with caries formation, one of the top ten health burdens affecting present day human populations. Overall, this thesis showcases the importance of expanding the ancient pathogenomics toolkit to include functional analytical approaches to study past pathogen genomes. Only in the light of archaeological contexts, will we gain insight into the emergence, ecology and long-term evolution of ancient pathogens
