Abstract: To explore the hybrid origin mechanism of Populus × jrtyschensis and P. canescens, the phylogenetic tree based on nuclear microsatellite markers (SSR) analysis was structured. The result show that the natural Populus populations can be classified into two groups, the one is Section Leuce, and the other is Section Aigeiros and Section Tacamahaca. In Section Leuce, significant genetic differentiation was found in the populations of Populus alba, P. canescens and P. tremula, especially the crossbreed Populus (P. canescens). A UPGMA phylogenetic tree shows that Section Aigeiros and Section Tacamahaca can be divided into two groups. Compared with Section Tacamahaca, the genetic differentiation in Section Aigeiros is more complex, and they can be further divided into four subgroups, including P. nigra, P. × jrtyschensis and their backcross progeny. It is confirmed that P. × jrtyschensis should be classified as Section Aigeiros based on their smaller genetic distance.