The first historically documented pandemic caused byYersinia pestisstarted as the Justinianic Plague in 541 within the Roman Empire and continued as the so-called First Pandemic until 750. Although palaeogenomic studies have previously identified the causative agent asY. pestis, little is known about the bacterium’s spread, diversity and genetic history over the course of the pandemic.To elucidate the microevolution of the bacterium during this time period, we screened human remains from 20 sites in Austria, Britain, Germany, France and Spain forY. pestisDNA and reconstructed six new genomes. We present a novel methodological approach assessing SNPs in ancient bacterial genomes, facilitating qualitative analyses of low coverage genomes from a metagenomic background. Phylogenetic analysis reveals the existence of previously undocumentedY. pestisdiversity during the 6th–7thcenturies, and provides evidence for the presence of multiple distinctY. pestisstrains in Europe. We offer genetic evidence for the presence of the Justinianic Plague in the British Isles, previously only hypothesized from ambiguous documentary accounts, as well as southern France and Spain, and that southern Germany seems to have been affected by at least two distinctY. pestisstrains. Four of the reported strains form a polytomy similar to others seen across theY. pestisphylogeny, associated with the Second and Third Pandemics. We identified a deletion of a 45 kb genomic region in the most recent First Pandemic strain affecting two virulence factors, intriguingly overlapping with a deletion found in 17th–18th-century genomes of the Second Pandemic.Significance StatementThe first historically reported pandemic attributed toYersinia pestisstarted with the Justinianic Plague (541–544) and continued for around 200 years as the so-called First Pandemic. To date, only oneY. pestisstrain from this pandemic has been reconstructed using ancient DNA. In this study, we present six new genomes from Britain, France, Germany and Spain, demonstrating the geographic range of plague during the First pandemic and showing microdiversity in the Early Medieval Period. Moreover, we detect similar genome decay during the First and Second Pandemic (17thto 18thcentury) that includes the same two virulence factors, thus providing an example of potential convergent evolution ofY. pestisduring large scale epidemics.