Pigs can play an important role in the genetic reassortment of influenza viruses and a reservoir for another lineage of influenza viruses that have the ability to reassort and be transmitted between species. On March and April 2006, novel H3N1 influenza A viruses were isolated from pigs with respiratory diseases at two different commercial swine farms in Korea. Genetic and phylogenetic analyses of the sequences of all eight viral RNA segments showed that the novel H3N1 swine influenza viruses were reassortants that acquired the hemagglutinin gene from an H3 human-like virus, and other genes from swine influenza viruses that are currently circulating in Korea. Serologic and virologic tests in the infected farms suggested that pig-to-pig and farm-to-farm transmissions occurred. Clinical signs in pigs and experimentally infected mice suggest the potential to transmit the virus between swine and other mammalian hosts. To our knowledge, this is the first report of the isolation of the swine H3N1 subtype from domestic pigs under field conditions in Korea. Further surveillance will be needed to determine whether this novel subtype will continue to circulate in the swine population.