Significance The bacterium Yersinia pestis has caused numerous historically documented outbreaks of plague and research using ancient DNA could demonstrate that it already affected human populations during the Neolithic. However, the pathogen’s genetic diversity, geographic spread, and transmission dynamics during this early period of Y. pestis evolution are largely unexplored. Here, we describe a set of ancient plague genomes up to 5,000 y old from across Eurasia. Our data demonstrate that two genetically distinct forms of Y. pestis evolved in parallel and were both distributed across vast geographic distances, potentially occupying different ecological niches. Interpreted within the archeological context, our results suggest that the spread of plague during this period was linked to increased human mobility and intensification of animal husbandry.