Abstract: To explore the replicating feature of H9 avian influenza virus (AIV) in chickens, A/Chicken/Guangdong/SS/94(H9N2) (SS) strain,which representing the earlier strain isolated in chickens in Guangdong province in 1994, and A/Chicken/Shanghai/F/98(H9N2) (F) strain, which was isolated in Shanghai during 1998 pandemic period, were selected as represent strains in this research. Three recombinants were constructed based on 6 genes from F strain and HA,NA genes from SS strain by reverse genetic technique. RF7/SSHA virus was composed of the HA gene from SS strain and the remaining seven genes from F strain, RF7/SSNA virus was composed of the NA gene derived from SS strain and the remaining seven genes from F strain, and RF6/SSHA/SSNA virus was composed of the HA and NA genes of SS strain and the remaining 6 genes from F strain. Then the replicating property of the five H9N2 viruses were compared in SPF chickens. Groups of three 4week old SPF chickens were inoculated with the AIVs by oral, tracheal and nasal routes. Virus replication was monitored 3 days after inoculation. Virus load in terms of ELD50/EID50 in trachea, lung and intestine were assayed 3 days after inoculation. The results showed that the replicating property of H9N2 subtype AIVs in chickens are determined by HA and NA genes mainly, and also related to internal genes. After 4 year’s adaption and gene evolution, F strain isolated in 1998 have stronger replication ability than earlier AIV strain in respiratory system, which lay the foundation of aerosol infection.