H9N2 avian influenza virus has been prevalent in poultry in many parts of the world since the 1990s and occasionally crosses the host barrier, transmitting to mammals, including humans. In recent years, these viruses have contributed genes to H5N1 and H7N9 influenza viruses, threatening the public health. To explore the molecular mechanism for the airborne transmission of H9N2 virus, we compared two genetically close strains isolated from chickens in 2001, A/chicken/Shanghai/7/2001(SH7) and A/chicken/Shanghai/14/2001(SH14). SH7 is airborne transmissible between chickens, whereas SH14 is not. We used reverse genetics and gene swapping to derive rSH7, rSH14 and a panel of reassortant viruses. Among the reassortant viruses, we identified segments HA and PA as governing the airborne transmission among chickens. In addition, the NP and NS genes also contributed to a lesser extent. Furthermore, the mutational analyses showed the transmissibility phenotype predominantly mapped to the HA and PA genes with HA-K363 and PA-L672 being important for airborne transmissibility among chickens. In addition, the viral infectivity and acid stability are related to the airborne transmissibility. Importantly, airborne transmission studies of 18 arbitrarily chosen H9N2 viruses from our collections confirmed the importance of both 363K in HA and 672L in PA in determining their transmissibilities. Our finding elucidates the genetic contributions to H9N2 transmissibility in chickens and highlights their importance in the prevalence in poultry.