Novel reassortant influenza A (H5N8) viruses are becoming a potential threat not only to the poultry industry but also to public health. Many molecular markers for pathogenicity in mammalian hosts have been identified in other H5 subtype avian influenza viruses (AIVs). However, the pathogenicity of H5N8 AIVs to mammals remains unclear. It is believed that selection of a pair of isolates with a similar genetic background but with different virulence to mammals is a prerequisite for studying the pathogenic mechanism of AIVs. Two avian-origin H5N8 isolates, A/goose/Eastern China/CZ/2013 (CZ13) and A/duck/ Eastern China /JY/2014 (JY14), which shared a similar genetic background (H5 clade 2.3.4.4) and amino acid substitutions that were shown previously to be molecular markers of pathogenicity, were used to determine their biological characteristics and pathogenicity. Hemagglutination assays using α-2,3-sialidase-treated goose red blood cells demonstrated that both viruses exhibited a dual-receptor-binding preference. Viral growth kinetics in vitro indicated that both viruses replicated to high titers in CEF cells (about 108.0 TCID50/mL). In MDCK cells, however, CZ13 replicated efficiently (107.0 TCID50/mL), while JY14 grew to peak titers below 104.0 TCID50/mL. Animal studies indicated that although both viruses were highly virulent in chickens, they exhibited significantly different virulence in mice. CZ13 was highly pathogenic (MLD50 = 101.6EID50), whereas JY14 had low virulence (MLD50 > 106.5 EID50). Therefore, this pair of viruses can be used to search for unknown molecular markers of virulence and to investigate specific pathogenic mechanisms in mice.