Low pathogenic (LP) H7N9 avian influenza virus (AIV) emerged in 2013 and had spread widely over several months in China, experienced a noteworthy reduction in isolation rate in poultry and human since 2017. Here, we examined the transmission of H7N9 viruses to better understand viral spread and dissemination mechanisms. Three out of four viruses (2013~2016) could transmit in chickens through direct contact, and airborne transmission was confirmed in the JT157 (2016) virus. However, we did not detect the transmission of the two 2017 viruses, WF69 and AH395, through either direct or airborne exposure. Molecular analysis of genome sequence of two viruses identified eleven mutations located in viral proteins (except for matrix protein), such as PA (K362R and S364N) and HA (D167N, H7 numbering), etc. We explored the genetic determinants that contributed to the difference in transmissibility of the viruses in chickens by generating a series of reassortants in the JT157 background. We found that the replacement of HA gene in JT157 by that of WF69 abrogated the airborne transmission in recipient chickens, whereas the combination of HA and PA replacement led to the loss of airborne and direct contact transmission. Failure with contact transmission of the viruses has been associated with the emergence of the mutations D167N in HA and K362R and S364N in PA. Furthermore, the HA D167N mutation significantly reduced viral attachment to chicken lung and trachea tissues, while mutations K362R and S364N in PA reduced the nuclear transport efficiency and the PA protein expression levels in both cytoplasm and nucleus of CEF cells. The D167N substitution in HA reduced the H7N9 viral acid stability and avian-like receptor binding, while enhanced human-like receptor binding. Further analysis revealed these mutants grew poorly in vitro and in vivo. To conclude, H7N9 AIVs that contain mutations in the HA and PA protein reduced the viral transmissibility in chicken, and may pose a reduced threat for poultry but remain a heightened public health risk.