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Swine are the main host of the H1N1 swine influenza virus (SIV), however, H1N1 can also infect humans and occasionally cause serious respiratory disease. To trace the evolution of the SIV in Guangdong, China, we performed an epidemic investigation during the period of 2016–2017. Nine H1N1 influenza viruses were isolated from swine nasal swabs. Antigenic analysis revealed that these viruses belonged to two distinct antigenic groups, represented by A/Swine/Guangdong/101/2016 and A/Swine/Guangdong/52/2017. Additionally, three genotypes, known as GD52/17-like, GD493/17-like and GD101/16-like, were identified by phylogenetic analysis. Importantly, the genotypes including a minimum of 4 pdm/09-origin internal genes have become prevalent in China in recent years. A total of 2966 swine serum samples were used to perform hemagglutination inhibition (HI) tests, and the results showed that the seroprevalence values of SW/GD/101/16 (32.2% in 2016, 32.1% in 2017) were significantly higher than the seroprevalence values of SW/GD/52/17 (18.0% in 2016, 16.7% in 2017). Our study showed that the three reassortant genotypes of H1N1 SIV currently circulating in China are stable, but H1N1pdm09 poses challenges to human health by the introduction of internal genes into these reassortant genotypes. Strengthening SIV surveillance is therefore critical for SIV control and minimizing its potential threat to public health. [ABSTRACT FROM AUTHOR]