Canine influenza (CI) is a contagious respiratory disease in dogs, which poses a threat to canine health. A safe, high-yield vaccine seed virus is critical for CI vaccine development. We developed a PR8-based reassortant H3N2 canine influenza virus (RT CIV) using the reverse genetic method and evaluated its yield in canine kidney epithelial (MDCK) cells, Vero cells, and specific pathogen-free (SPF) chicken embryos. Mice and dogs were infected with RT CIV, and the pathogenicity was evaluated. The viral titers of RT CIV increased in MDCK cells, Vero cells, and SPF chicken embryos; the HA yield in SPF chicken embryos increased 4-fold. However, RT CIV was not lethal to mice, and it showed similar virulence as wild-type CIV. RT CIV also showed minimal pathogenicity in dogs, which manifested as mild fever and rhinorrhea for the first two days post-infection. Thus, RT CIV carrying the internal gene cassette from PR8 showed almost no pathogenicity in dogs. And the reassortant virus inactivated vaccine could provide complete protection against H3N2 CIV. To our knowledge, this is the first report on the pathogenicity of PR8-based reassortant H3N2 CIV in dogs. These studies are relevant for developing a high-yield and safe CI vaccine.