Swine influenza is an acute respiratory disease of swine caused by type A influenza viruses. Before 1998, mainly "classical" HIN1 swine influenza viruses (SIVs) were isolated from swine in the United States. Since then, antigenically distinct reassortant H3 and H1 SIVs have been identified as causative agents of respiratory disease in pigs on US farms. Improvement in SIV diagnostics is needed in light of the recently observed rapid evolution of H1 and H3 SIVs and their zoonotic potential. To address this need, real-time reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of SIVs were developed. A highly sensitive matrix (M) gene-based RT-PCR assay that is able to detect both the H1 and H3 subtypes of SIVs, with a sensitivity per reaction of approximately 2 copies of in vitro-generated M-specific negative-sense RNA molecules and approximately 0.05 TCID50 in lung lavage of experimentally SIV-infected pigs, was established. This RT-PCR assay can be performed within a few hours and showed a sensitivity of 94% and a specificity of 85% when compared with virus isolation. In addition, H1-, H3-, N1-, and N2-specific primer and probe sets were designed for use in the differentiation of different SIV subtypes. The hemagglutinin (H)- and neuraminidase (N)-specific primer and probe sets were less sensitive than the M-specific assay, although they were found to be specific for their respective viral genes and able to distinguish between their respective SIV subtypes.