The H3N2 canine influenza virus (CIV), which originated from avian influenza viruses and has circulated in dogs for over two decades, remains poorly understood in terms of its host adaptation mechanisms and cellular tropism. In this study, we isolated two phenotypically distinct H3N2 CIV strains (CX3 and CX12) from a canine respiratory disease outbreak. Although both were isolated using MDCK cells, CX3 showed higher replication efficiency and plaque-forming ability in MDCK cells, whereas CX12 replicated more efficiently in chicken embryos. Using reverse genetics, we identified the PB1 gene as a critical enhancer of viral polymerase activity and the HA gene as a key contributor to viral stability, both of which collectively mediate cell-preferred replication. These findings provide mechanistic insights into H3N2 CIV host adaptation and offer a foundation for future vaccine development.