Seasonal influenza is caused by two subtypes of influenza A virus (A/H1N1 and A/H3N2) and two lineages of influenza B viruses (B/Victoria-lineage and B/Yamagata-lineage). Seasonal influenza viruses replicate efficiently in the human upper respiratory tract, where the temperature is 33 °C. In this study, we investigated the susceptibility of seasonal influenza A and B viruses to different temperatures. We examined the differences in viral replication efficiency inside cultured cells and in infectious titre outside cultured cells at different temperatures (i.e., 33 °C, 37 °C, and 39 °C). We found that there were differences in temperature sensitivity between influenza A and B viruses, with influenza B viruses being more temperature sensitive. In addition, we found that cells cultured at 39 °C and infected with influenza B virus showed decreased expression of HA protein with receptor-binding activity on the cell surface. Our findings contribute to our understanding of the properties of seasonal influenza viruses.