Canine herpesvirus (CHV) is a member of the alphaherpesvirus subfamily, which can cause severe hemorrhagic diseases in neonatal pups as well as mild or subclinical respiratory infections in adult dogs. We examined the effects of cold stress on disease progression of CHV, an alphaherpesvirus, in neonatal puppies. Eight puppies were challenged intranasally with CHV suspension and divided into a cold stress treatment group and a hyperthermal group. Four pups were left uninoculated as controls and divided into cold and hyperthermal groups. In the challenged cold treatment group, all pups showed CHV-related disease within 5 days; pathological changes were observed in organs of puppies showing clinical symptoms. Grossly, numerous petechial red foci were scattered throughout lungs, kidneys, livers, and intestines of all CHV-infected puppies exposed to cold stress. Most puppies showed typical clinical signs and macroscopic lesions, and CHV infection was confirmed by isolation of the virus. However, in the challenged hyperthermal group, only one of the pups showed mild symptoms of CHV-induced disease. None of the puppies in the uninoculated group showed abnormal signs, although they were exposed to cold stress. These findings indicate that cold stress can cause rapid disease progression of CHV, an alphaherpesvirus.