Recently, canine influenza virus H3N2 (CIV H3N2) has circulated continuously in the dog populations of Asia and the United States (US). As humans have close contact with pet dogs, the circulation of CIV H3N2 is a cause for concern. Previous studies have reported that the E627K and D701N substitutions in the PB2 subunit enhanced viral pathogenicity to mammals in various influenza viruses. However, how the E627K and D701N substitutions in the PB2 subunit might affect the virulence of CIV H3N2 is unclear. Here, we constructed recombinant viruses by introducing E627K or D701N into the PB2 gene in the genetic background of A/Canine/Guangdong/02/2011H3N2 using a reverse-genetic system. The results showed that the E627K or D701N substitutions in the PB2 subunit of CIV H3N2 enhanced polymerase activity, but these substitutions did not impact viral pathogenicity in mice or beagles.