The origin of the medieval Black Death pandemic ( ad 1346–1353) has been a topic of continuous investigation because of the pandemic’s extensive demographic impact and long-lasting consequences 1, 2 . Until now, the most debated archaeological evidence potentially associated with the pandemic’s initiation derives from cemeteries located near Lake Issyk-Kul of modern-day Kyrgyzstan 1, 3– 9 . These sites are thought to have housed victims of a fourteenth-century epidemic as tombstone inscriptions directly dated to 1338–1339 state ‘pestilence’ as the cause of death for the buried individuals 9 . Here we report ancient DNA data from seven individuals exhumed from two of these cemeteries, Kara-Djigach and Burana. Our synthesis of archaeological, historical and ancient genomic data shows a clear involvement of the plague bacterium Yersinia pestis in this epidemic event. Two reconstructed ancient Y. pestis genomes represent a single strain and are identified as the most recent common ancestor of a major diversification commonly associated with the pandemic’s emergence, here dated to the first half of the fourteenth century. Comparisons with present-day diversity from Y. pestis reservoirs in the extended Tian Shan region support a local emergence of the recovered ancient strain. Through multiple lines of evidence, our data support an early fourteenth-century source of the second plague pandemic in central Eurasia.