Wild birds are considered to be the natural reservoir hosts of avian influenza viruses (AIVs). Wild bird-origin AIVs may spill over into new hosts and overcome species barriers after evolutionary adaptation. H13N8 AIVs used to be considered primarily circulated in multispecies gulls but have recently been shown to possess cross-species infectivity. In this study, we analyzed the genetic changes that occurred in the process of the evolution of H13 AIVs. Phylogenetic analysis revealed that H13 AIVs underwent complex reassortment events. Based on the full genomic diversity, we divided H13 AIVs into 81 genotypes. Reassortment experiments indicated that basic polymerase 2 (PB2) and nucleoprotein (NP) genes of the H9N2 AIV significantly enhanced the polymerase activity of the H13N8 AIV. Using the replication-incompetent virus screening system, we identified two mutations, PB2-I76T and PB2-I559T, which could enhance the polymerase activity of the H13N8 AIV in mammalian cells. Notably, these mutations had been acquired by circulating H13N8 AIVs in 2015. These findings suggest that H13N8 AIVs are about to cross the host barrier. Occasional genetic reassortments with other AIVs and natural mutation events could promote this process. It is imperative to intensify monitoring efforts for H13N8 AIVs.