Zhimin Wana,b,c,d*, Wenjie Jianga,b,c,d*, Xudong Caoa,b,c,d, Xingyao Guoa,b,c,d, Xinyan Hea,b,c,d, Jianjun Zhange,Xinran Chua,b,c,d, Xinyi Jia,b,c,d, Yu Liua,b,c,d, Xuefeng Yina,b,c,d, Zhehong Zhaoa,b,c,d, Jixiang Wanga,b,c,d,Wei Gaoa,b,c,d, Quan Xiea,b,c,d, Tuofan Lia,b,c,d, Hongxia Shaoa,b,c, Aijian Qina,b,c, Yuhai Bif,g,h andJianqiang Yea,b,ca In recent decades, multiple novel reassortant avian influenza viruses (AIVs) carrying internal genes derived from H9N2 viruses have emerged in poultry in China and have repeatedly caused human infections, highlighting the pivotal role of the H9N2 internal genes cassette in facilitating cross-species transmission. In this study, five H6N1 and one H6N2 AIVs were isolated from chickens and Houdan chickens exhibiting respiratory symptoms. Genetic analyses revealed that the hemagglutinin (HA) genes of these H6 AIVs belonged to the ST339-like lineage and originated from H6 AIVs circulating in domestic geese in China, whereas the neuraminidase (NA) genes were derived from H9N2 or H5N1 AIVs, respectively. Notably, all six internal genes of these H6 isolates were derived from the G57 genotype H9N2 virus. In vitro studies demonstrated that these H6 AIVs replicated effectively in both avian and mammalian cells. Discussion The continuous emergence of the novel reassortant AIVs poses a great threat to both poultry industry and public health. In this study, we identified and characterized six...