Despite the growing number of recent studies on genome-wide divergence during speciation, the genetic basis and mechanisms of genomic divergence and speciation are still incompletely understood. In 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 also cause genome heterogeneity and speciation. Based on whole genome re-sequencing data from 27 Populus alba and 28 P. adenopoda individuals, we explored the reasons for the heterogeneous differentiation of genomes of these two closely related species. The results showed that 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, which caused the two species begin to diverge and eventually led to speciation. In highly differentiated regions, neutrality tests (Tajima’s D and Fay & Wu’s H) of these regions revealed no difference while the absolute divergence (dxy) were significantly higher than genome background, which indicates that barriers to gene flow rather than natural selection played a major role in maintaining genomic heterogeneous divergence and reproductive isolation, which is the most important condition for speciation. We further found that some genes related to reproduction may be involved in explaining the reproductive isolation of the two species.