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• Chickens are susceptible to H5N6 avian influenza viruses of different origins. • The H5N6 viruses were effectively transmitted in chickens but caused different mortality rates. • All tested immune genes were markedly upregulated within 24 hpi in virus-infected chickens. The H5N6 highly pathogenic avian influenza virus (HPAIV) has been circulating in China since 2013. In this report, we describe our recent chicken experimental studies investigating the pathogenicity and transmission of four H5N6 HPAIV field strains of different origins (GS39, CK44, DK47 and CK74) and the host immune responses. Four-week-old specific-pathogen-free chickens were inoculated intranasally with one of the four H5N6 HPAIV strains (one strain per group). Among the contact chickens, the GS39 and CK74 strains caused 100 % mortality, the CK44 strain caused 80 % mortality, and the DK47 strain caused 40 % mortality. The viruses were effectively replicated in multiple tissues of the inoculated chickens, in which high viral titers were detected in virus-infected tissues, and significantly upregulated expression of immune-related genes was found in the infected chickens at 24 hpi. The chicken serum antibody levels increased from 5log2 at 7 dpe to 7.67−8log2 at 14 dpe. The major histocompatibility complex molecules were upregulated 21.22- to 32.98-fold in lungs and 5.10- to 18.47-fold in spleens. In summary, H5N6 viruses can replicate within chickens and be effectively transmitted between chickens. Our study contributes to further understanding the pathogenesis of clade 2.3.4.4 H5N6 avian influenza viruses in chickens. [ABSTRACT FROM AUTHOR]