SignificanceWhile our knowledge of modern plague reservoirs and their hosts is extensive, we have little to no knowledge about the origin of the Medieval plague pandemics or the routes of transmission involved in their spread. Prior genomic data provide a patchy low-resolution picture of the transmission dynamics involved during the Second Plague Pandemic, with only five distinct genomes. We have reevaluated all Medieval strains under the light of archaeological and historical evidence to carefully discuss the involvement of different transmission routes during the Second Plague Pandemic. Our interpretation showcases the importance of trade routes and human movements and further supports the identification of Yersinia pestis as the pathogenic agent of the so-called pestis secunda (1357–1366).

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