The prevention of infectious disease via prophylactic immunization is a mainstay of global public health efforts. Vaccine design would be facilitated by a better understanding of the type and durability of immune responses generated by different vaccine vectors. We report here the results of a comparative immunogenicity trial of six different vaccine vectors expressing the same insert antigen, cowpox virus B5 (CPXV-B5). Of those vectors tested, recombinant adenovirus (rAd5) was the most immunogenic, inducing the highest titer anti-B5 antibodies and conferring protection from sublethal vaccinia virus challenge in mice after a single immunization. We tested select heterologous prime-boost combinations and identified recombinant vesicular stomatitis virus (rVSV) and recombinant Venezuelan equine encephalitis virus replicons (VRP) as the most synergistic regimen. Comparative data such as those presented here are critical to efforts to generate protective vaccines for emerging infectious diseases as well as for biothreat agents.