Abstract: Members of the genus Juglans (walnuts, family Juglandaceae) are typical Tertiary relict tree species. They are widely distributed in China and are well known for their edible nuts and high‐quality wood. The genetic structure and historical gene flow patterns of Chinese Juglans still need to be resolved. Here, we collected samples from a total of 2242 trees from 142 populations of three walnut species (Juglans regia L., Juglans sigillata Dode, and Juglans mandshurica Maxim.) and a hybrid taxon Juglans × hopeiensis. These samples were distributed throughout China. We analyzed population genetic structure, interspecific relationships, lineage differentiation, and speciation using 21 EST‐SSR genetic markers. All populations of J. regia and J. sigillata clustered into one lineage, corresponding to the Juglans sect. Dioscaryon, and all J. mandshurica and Juglans × hopeiensis populations (section Cardiocaryon) clustered into a second lineage. For J. mandshurica, there was an obvious north-south genetic cline. Interestingly, except for seven populations of the hybrid Juglans × hopeiensis, there was almost no gene flow between the populations of section Juglans/Dioscaryon and section Cardiocaryon. A DIYABC analysis showed that J. regia and J. mandshurica differentiated during the Oligocene. Juglans sigillata originated during the Pliocene to the Pleistocene. Subsequently, during the Middle Pleistocene, J. regia and J. mandshurica hybridized within a narrow zone to produce Juglans × hopeiensis. These results demonstrate the impact of repeated turbulent climate changes in the Quaternary on the evolutionary history of Tertiary relict plants.

Key words: comparative phylogeography, evolutionary history, gene introgression,  Juglans,  Juglans mandshurica,  Juglans regia