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Background: The fifth wave of H7N9 avian influenza virus caused a large number of human infections and a large number of poultry deaths in China. Since September 2017, mainland China has begun to vaccinate poultry with H5 + H7 avian influenza vaccine. We investigated the avian influenza virus infections in different types of live poultry markets and samples before and after genotype H5 + H7 vaccination in Nanchang, and analyzed the changes of the HA subtypes of AIVs. Methods: From 2016 to 2019, we monitored different live poultry markets and collected specimens, using real-time reverse transcription polymerase chain reaction (RT-PCR) technology to detect the nucleic acid of type A avian influenza virus in the samples. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. The χ2 test was used to compare the differences in the separation rates of different avian influenza subtypes. Results: We analyzed 5,196 samples collected before and after vaccination and found that the infection rate of AIV in wholesale market (21.73%) was lower than that in retail market (24.74%) (P < 0.05). Among all the samples, the positive rate of sewage samples (33.90%) was the highest (P < 0.001). After vaccination, the positive rate of H5 and H7 subtypes decreased, and the positive rate of H9 subtype and untypable HA type increased significantly (P < 0.001). The positive rates of H9 subtype in different types of LPMs and different types of samples increased significantly (P < 0.01), and the positive rates of untypable HA type increased significantly in all environmental samples (P < 0.05). Conclusions: Since vaccination, the positive rates of H5 and H7 subtypes have decreased, but the positive rates of H9 subtypes have increased to varying degrees in different testing locations and all samples. This results show that the government should establish more complete measures to achieve long-term control of the avian influenza virus. [ABSTRACT FROM AUTHOR]