Duck viral enteritis is an acute, contagious infection of Anatidae family members. The disease is caused by Anatid herpesvirus 1 (AnHV-1). The infection of AnHV-1 is controlled by vaccination to the flock with chick embryo adapted attenuated vaccine in developed countries. However, its economic impact in developing countries is substantial and there is a need to understand the cell culture spectrum of the virus to produce its vaccine on a mass scale. In the present study, the permissivity of AnHV-1 for different cells was analyzed. The AnHV-1 showed enhanced replication following its serial passage in CEF, DF-1, Vero, MDCK, and QT-35 cells. The characteristic cytopathic effect (CPE) of rounding and clumping of cells were observed in CEF, DF-1, Vero, and QT-35 cell lines. The infectivity and viral replication were highest in CEF, DF-1, Vero, and QT-35 cells. In contrast, the results suggested that MDCK cells are less permissive for AnHV-1 infection with negligible CPE and reduced viral replication. Heterologous cell culture systems other than chicken embryo fibroblasts to adapted live vaccine viruses will provide a system devoid of other avian infectious agents. Moreover, it can be used for the propagation and cultivation of AnHV-1 vaccine strain for developing cell culture-based vaccines with high titer and could be an economical alternative for the existing options.