Whole animal imaging allows viral replication and localization to be monitored in intact animals, which provides significant advantages for determining viral and host factors that determine pathogenesis. To investigate effects of interferons on spatial and temporal progression of vaccinia infection, we generated recombinant viruses that express firefly luciferase or a monomeric orange fluorescent protein. These viruses allow vaccinia infection to be monitored with bioluminescence or fluorescence imaging, respectively. The recombinant viruses were not attenuated in vitro or in vivo relative to a control WR virus. In cell culture, reporters could be detected readily by 4 h post-infection, showing that these viruses can be used as early markers of infection. The magnitude of firefly luciferase activity measured with bioluminescence imaging in vitro and in vivo correlated directly with increasing titers of vaccinia virus, validating imaging data as a marker of viral infection. Replication of vaccinia was significantly greater in mice lacking receptors for type I interferons (IFN I R^−/−) compared with wild-type mice, although both genotypes of mice developed focal infections in lungs and brain after intranasal inoculation. IFN I R^−/− mice had greater dissemination of virus to liver and spleen than wild-type animals even when mortality occurred at the same time point after infection. Protective effects of type I interferons were mediated primarily through parenchymal cells rather than hematopoietic cells as analyzed by bone marrow transplant experiments. Collectively, our data define a new function for type I interferon signaling in systemic dissemination of vaccinia and validate these reporter viruses for studies of pathogenesis.