Two non-radioactive probes using digoxigenin or biotin were developed for detecting canine herpesvirus (CHV) and compared for their sensitivities by in situ hybridization (ISH) in formalin fixed, paraffin embedded sections, which has been used routinely in veterinary fields. Sections of the CHV-infected cell preparation were subjected to several different ISH protocols using digoxigenin- or biotin-labeled probe respectively. Results were compared for the hybridization and background signal intensities. The best result was obtained by the optimized ISH protocol using digoxigenin-labeled probe for detection of CHV DNA. The optimized ISH assay, which developed in this study, may be a valid tool for the study of pathogenesis and diagnosis of CHV infection.