The Matrix (M) genes of 6 H9N2 subtype avian Influenza Viruses (AIVs)which isolated from chickens in Shandong in 2009 , were sequenced and molecular evolutionarily analyzed in this study.Homology of nucleotide sequences in the M gene of 6 isolates was 95.1–99.4%. The identity among the M1 protein was 97.6–99.6% while the M2 protein was 95.1–100%. Phylogenetic analysis showed that all of the tested M genes belong to the A/Quail/Hong- Kong/G1/97 (G1)-like lineage. An analysis of the viral amino acid sequence of M2 protein has revealed substitution of S31N in two isolates, which is the molecular characterization of amantadine resistance in AIVs. The BALB/c mice aged 6 weeks with 106 EID50 H9N2, lost weight progressively, and some died within 7 days of infection. Results of this study suggest that long-term monitoring should be continued to track the transmission and evolution of H9N2 AIVs in chickens in China.