【Objective】To confirm the effect of temperature on proliferation in vitro and pathogenicity of Cyprinid herpesvirus 3 (CyHV-3).【Method】CyHV-3-EGFP strain was used in this study. Viral titer was measured by the indirect immunofluorescence staining assay. The proliferation of virus in vitro was observed using a fluorescence microscope. The thermal stability, and the effect of temperature on proliferation in vitro and pathogenicity in vivo of virus were studied.【Result】CyHV-3 had good thermal stability at 4-36 ℃. The viability of CyHV-3 decreased with the increase of temperature when the temperature exceeded 36 ℃. The virus viability was completely lost at 50 ℃. The infection temperature had little effect on the viability of CyHV-3, and the virus showed infectivity at both high (37 ℃) and low (4 ℃) temperatures. However, incubation temperature significantly affected the virus viability. CyHV-3 proliferation capacity decreased or even disappeared when the temperature exceeded 30 ℃. After CyHV-3 infection, the mortality rates for the carps at low temperature (15 ℃) and room temperature (25 ℃) were 26.67% and 73.33% respectively, and the mortality rates for the carps at high temperature (30 ℃) and in the control group with no virus infection were 0.【Conclusion】The results confirm that CyHV-3 can proliferate and has pathogenicity at low temperature, which explain the phenomenon that the outbreak and rapid spread of KHVD in recent years are no longer restricted in spring and autumn seasons.