To understand the genetic evolution characteristics of H6N3 subtype avian influenza virus (AIV), throat swabs and cloacal swabs were collected from live poultry markets in Guangdong and Hubei province in 2023 to isolate and identify the viruses. The whole genome of H6N3 subtype AIV were sequenced, and the genetic evolution and special molecular markers were analyzed. The results showed that 5 strains of H6N3 subtype AIV were isolated, which were novel reassortment viruses that could be divided into 2 genotypes. The 4 strains isolated from Hubei province belonged to genotype 1, the NA gene of which constituted a single branch,
and the other gene segments were closely related to H3N2, H3N3, H3N8 and H6N6 subtypes AIV isolated from poultry in China. One strain isolated from Guangdong province belonged to genotype 2, and the genes of this virus were highly homologous with different subtypes AIV from wild birds. There was only one basic amino acid in the cleavage site of HA protein of 5 isolates, which were consistent with the low pathogenic avian influenza virus (LPAIV). The length of the NA gene of 4 strains isolated from Hubei province was only 1 365 bp, with a deletion in the stalk of NA. The A138S substitutions was found in HA protein of some viruses, and the I292V was found in the PB2 of 1 isolate. In conclusion, the 5 strains of H6N3 subtype AIV were novel reassortment viruses and some strains had acquired mammalian adaptive substitutions, and epidemiological surveillance of H6 subtype AIV should be intensified.