RepliVax, a novel replication-defective vaccine platform has recently been described as a suitable means of generating potent vaccines targeting flaviviruses. In this study, we directly compared attenuation, immunogenicity and efficacy of several prototype RepliVax constructs to available, well characterized live attenuated (LAV) and inactivated (INV) flavivirus vaccine controls in mice and hamsters. Other important aspects of general mechanisms and properties of RepliVax vaccines were also studied. The prototypes were found to be nonpathogenic in sensitive suckling mouse neurovirulence tests, and highly immunogenic and efficacious in mice and hamsters, with evidence that immunogenicity can be comparable to LAV controls in terms of both magnitude and durability of response. Our data also suggest that choice of inoculation route can be beneficial for maximizing RepliVax immunogenicity. Additionally, different vaccine constructs can be administered as cocktail formulations without compromising immunogenicity of individual components. RepliVax constructs were determined to induce a Th1 biased immune response, similar to LAVs, and different from INV inducing a Th2 type response. The results presented validate the utility of the RepliVax platform for development of novel flavivirus vaccines. © 2011 Elsevier Ltd.