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Wheat leaf rust is caused by Puccinia triticina (Pt), leading to serious wheat yield loss in the world. To study the population structure and reveal the transmission routes of Pt in eastern China, leaf samples were collected from the main wheat-producing areas from April to June 2020. Total of 372 Pt strains were amplified by 13 SSR makers and a high level of genetic diversity was revealed with 289 multi-locus genotypes (MLG) identified. STRUCTURE analysis suggests that all Pt strains were assigned to 3 clusters, and 11 populations were further defined by considering geographic locations. All 55 pairwise populations had number of migration (Nm) values > 1, indicating moderate genetic differentiation and frequent exchanges among populations. The genetic structure was significant different among populations in the northern and southern regions bounded by the Qinling Mountains-Huaihe River line. Pt strains in the southern regions, such as Jiangsu, Anhui and Zhejiang provinces, had higher level of genetic diversity and genetic variation, and Jiangsu might play an important role in the epidemic and population structure of Pt. Both genetic communication and horizontal wind field analyses showed that Pt had higher level of gene flow from the southern to northern regions than that of the reverse direction. The demonstrated genetic structure and dispersal route of Chinese eastern Pt populations would provide valuable information for epidemiological studies and disease control. [ABSTRACT FROM AUTHOR]