【Background】H6 avian influenza virus (AIV) is widely prevalent in southern China, which is one of the most common subtypes of AIVs circulating in poultry in China. H6N1 AIVs frequently undergo gene reassortment events with other wild bird-origin virus, which can be a donor to provide internal gene segments to highly pathogenic AIVs, which may lead to the emergence of novel virus and threaten human health.【Objective】The aim of this study was to investigate the evolution and biological characteristics of H6N1 AIVs in China, so as to provide valuable data for the prevention and control of avian influenza in China.【Method】From 2019 to 2022, cloacal and throat swabs were collected from live poultry markets and breeding farms across 25 provinces or autonomous regions in China. 7 H6N1 viruses were isolated by inoculating into chicken embryos. The complete genome sequences were determined, followed by analyzing their phylogenetic relationships, receptor binding properties, as well as replication in SPF chickens and BALB/c mice.【Result】The phylogenetic analysis revealed that the genes of the 7 H6N1 AIVs were highly homologous to those of wild bird-origin viruses from North America and Southeast Asia, indicating a complex genetic origin with significant genetic diversity. According to the Bayesian evolution analysis, the HA gene of H6 subtype AIVs had many times of intercontinental transmission in the history, and the Eurasian lineage strains also had a long time of circulation in North America. The HA gene of one strain of the virus was highly homologous to that of the North American strain. Based on the results of Bayesian evolutionary analysis, it was hypothesized that the virus was introduced to China via wild birds after undergoing complex genetic reassortment in wild birds. Analysis of specific amino acid sites revealed that the cleavage site of HA protein was PQIETR↓GLF, which was the signature of low pathogenic AIVs. In addition, one other virus had a Y52H mutation in the NP protein, which was critical in BTN3A3 evasion. Receptor binding analysis demonstrated that some of the H6N1 AIVs bound to both avian-type receptor and human-type receptor, however, their affinity towards human receptors was weaker compared with avian receptors. Infectivity experiments on SPF chickens indicated that flocks was still able to shed virus up through oropharyngeal and cloacal routes after infection with A/chicken/Jiangxi/S40445/2019(H6N1) and that the virus could be transmitted via contact within flocks. Only a few chickens infected with A/duck/Jiangxi/S10941/2019(H6N1) shed virus through their oropharyngeal tract, and the virus could not be transmitted via contact among chickens. The infectivity experiments conducted on mice showed that H6N1 subtype AIVs could replicate in the respiratory organs of infected mice without prior adaptation, but display low pathogenicity levels in mice.【Conclusion】Most of the genes of H6N1 subtype AIV isolated in China from 2019 to 2022 were derived from wild bird-origin viruses, and those migratory birds could introduce the viruses into China via the East Asia-Australasian migratory flyway. Some of the H6 AIVs bound to human-type receptors and replicate in the respiratory organs of mice suggested that H6N1 viruses posed a potential threat to human health.