During recent canine influenza surveillance in South Korea, a novel canine influenza virus (CIV) H3N1 which was a putative reassortant between pandemic H1N1 2009 and H3N2 canine influenza viruses was isolated. Genetic analysis for 8 genomes of the influenza virus revealed that novel H3N1 isolate presented high similarities (99.1-99.8%) with pandemic influenza H1N1 except for the hemagglutinin (HA) gene. The HA gene nucleotide sequences of the novel CIV H3N1 was similar (99%) to that of CIV H3N2 isolated in Korea and China. Through the inoculation of the novel CIV H3N1 virus, the novel isolate could induce the clinical signs including fever, cough, and nasal discharges and also presented gross lesions in lungs and histopathologic changes.