AbstractIn most species, natural selection plays a key role in genomic heterogeneous divergence. Additionally, barriers to gene flow, such as chromosomal rearrangements or gene incompatibilities, can cause genome heterogeneity. We used genome-wide re-sequencing data from 27 Populus alba and 28 P. adenopoda individuals to explore the causes of genomic heterogeneous differentiation in these two closely related species. In highly differentiated regions, neutrality tests (Tajima’s D and Fay & Wu’s H) revealed no difference while the absolute divergence (dxy) were significantly higher than genome background, which indicates that natural selection did not play a major role but barriers to gene flow play an important role in generating genomic heterogeneous divergence and reproductive isolation. The two species diverged ∼5-10 million years ago (Mya), when the Qinghai-Tibet Plateau reached a certain height and the inland climate of the Asian continent became arid. We further found some genes that are related to reproduction.