An overall 8,427 total of blood samples collected from growing to finishing pigs were submitted for diagnosis of swine influenza virus infection between January 2002 and December 2006. Sera from 2002 to 2005 were examined for antibodies against four different swine influenza subtypes using the hemagglutination inhibition test to investigate seroprevalence rates by natural infection in Korean swine herds while nasal swabs and lung tissue samples were used for viral isolation. The natural infection rate of subtypes H1, H3, and the dual positive were 41.5%, 3.6%, and 0.9%, respectively. Of the 687 swine nasal swab specimens collected from pigs with respiratory diseases, forty swine influenza viruses were isolated and subtyped as H1N1 (2 isolates), H1N2 (28 isolates), or H3N2 (10 isolates) by multiplex RT-PCR and sequencing. Although all three subtypes are currently co-circulating in South Korea, the H1N2 subtype was the most commonly isolated in this study which are almost monophyletic in all gene segments. In contrast, while earlier H3N2 isolates reflect the three cluster groups in the United States, more recent isolates obtained after the national vaccination program in October 2005 showed two distinguishable lineages. The first group appears to be derived from serogroup III US H3N2 SIVs. The other group are new reassortant viruses of this subtype where the HA genes originate from an early human-like isolate (New York/647/95) while the remaining genes are of swine-like lineage. Phylogenetic analyses indicate that the H1 and H3 viruses are actively evolving in Korean swine herds by multiple, independent reassortment events.