The recent concurrent emergence of H5N1, H5N6, and H5N8 avian influenza viruses (AIVs) has caused significant avian mortality globally. Since 2020, frequent human-animal interactions have been documented. To gain insight into the novel H5 subtype AIVs (i.e., H5N1, H5N6 and H5N8), we conducted a comparative analysis on phylogenetic evolutionary and biological properties of H5 subtype AIVs strains isolated from China between January 2021 and September 2022. Phylogenetic analysis revealed that the 41 H5Nx strains belonged to clade 2.3.4.4b, with 13 related to H5N1, 19 to H5N6, and 9 to H5N8. The genetic relatedness analysis based on global 2.3.4.4b viruses showed that all the viruses described in this study was likely originated from H5N8, exhibiting a heterogeneous evolutionary history between H5N1 and H5N6 during 2015–2022 worldwide. In this context, we further estimated that H5N1, characterized by higher evolutionary rates in 2021–2022 and more sites under positive selection pressure in 2015–2022. The antigenic profiles of novel H5N1 and H5N6 exhibited notable variations. Further hemagglutination inhibition assay suggest that some A(H5N1) viruses may be antigenically distinct from the circulating H5N6 and H5N8 strains. Mammalian challenge assays demonstrated that the H5N8 virus (21GD001_H5N8) displayed the highest pathogenicity in mice, followed by the H5N1 virus (B1557_H5N1) and then the H5N6 virus (220086_H5N6), suggesting a heterogeneous virulence profile of H5 AIVs in the mammalian hosts. Based on the above results, we consider that A(H5N1) viruses have a higher risk of emergence in the future. Collectively, these findings unveil a new landscape of different evolutionary history and biological characteristics of novel H5 AIVs in clade 2.3.4.4b, contributing to a better understanding for designing more effective strategies for the prevention and control of novel H5 AIVs.