Objective: :Transmission of highly pathogenic H5N1 influenza viruses from poultry to humans and from humans to humans is currently inefficient. However, when such transmission occurs, the case-fatality ratio is high. Materials and Methods: 1. Reassortant viruses were generated with an eight-plasmid reverse genetics system. 2. A thin-layer chromatography (TLC)/virus-binding assay and an enzyme-linked immunosorbent assay(ELISA)/virus-binding assay were used to determine the binding specificity of H5N1 subtype viruses to SPG as receptor substrate. 3. Cell infection experiment Results: Fifty-five H5N1 isolates from poultry and humans in recent years could be divided into three groups based on receptor binding specificity for the avian-type receptor, the human-type receptor and both types of receptors. Analysis of hemagglutinin (HA) and neuraminidase (NA) sequences of these viruses revealed motifs associated with the receptor binding profile. We determined that glycosylation of HA and length of NA stalk influenced the receptor binding specificities of H5N1 viruses using a reverse genetic system. The viruses having two glycosylation at positions 158 and 169 in HA lacked the binding activities to human-type receptor. Data of their binding specificity and their replication in different type cells indicates the length of NA stalk was associated with the receptor binding profile, further act on the replication of viruses. Viruses which match with HA having two glycosylation sites and short-stalked NA bound preferentially to avian-type receptor and adapted to poultry. Viruses which match with HA lacking glycosylation at 158 position and short-stalked NA bound both avian- and human-type receptors. Conclusion: We also found two critical factors favoring H5N1 infection from humans to humans: no glycosylation site at position 158 of HA, and NA with a full-length stalk. Such viruses might be more likely to be transmitted from humans to humans. These features are important molecular markers of H5N1 viruses with increased pandemic potential.