Previous studies have shown that chimeric bat influenza viruses can be generated by reverse genetic system. However, the roles of the surface or internal genes of chimeric bat influenza viruses in viral replication and virulence in different host species were still not completely understood. In this study, we generated a chimeric H9N2 bat virus with both HA and NA surface genes from the avian A2093/H9N2 virus and compared its replication and virulence with the chimeric H1N1 bat virus with both HA and NA from the PR8/H1N1 virus in vitro and in mice. The chimeric H1N1 virus showed significantly higher replication in mammalian and avian cells and significantly higher virulence in mice than the chimeric H9N2 virus. Moreover, the chimeric H9N2 virus with the bat influenza internal M gene showed a higher replication in mammalian cells than in avian cells. While the chimeric H9N2 virus with the avian-origin viral M gene displayed a higher replication than that with the bat influenza M gene in avian cells, which likely resulted from increased receptor binding ability to α 2,3 sialic acid linked glycans of the former virus. Our study indicates that bat influenza internal genes are permissive in both mammalian and avian cells, and the bat influenza internal M gene shows more compatibility in mammals than in the avian host. Although the surface genes play more critical roles for viral replication in different host substrates, influenza M gene also potentially impacts on replication, virulence and host tropism.