The immunogenicity of adenovirus serotype 5 (Ad5) vectors has been shown to be suppressed by neutralizing antibodies (NAbs) directed primarily against the hexon hypervariable regions (HVRs). We previously reported that replacing all seven HVRs with those from the rare serotype virus Ad48 resulted in a chimeric Ad5HVR48(1-7) vector that largely evaded preexisting Ad5 immunity in mice and rhesus monkeys. In this study, we evaluated the extent to which Ad5-specific NAbs are directed against various HVRs. We constructed partial HVR-chimeric Ad5 vectors with only a subset of HVRs exchanged, and we utilized these vectors in both NAb assays and murine immunogenicity studies with and without baseline Ad5 immunity. Our results demonstrate that Ad5-specific NAbs target multiple HVRs, suggesting that replacing all HVRs is required to optimize evasion of anti-Ad5 immunity. These data have important implications for the development of novel vectors for both vaccines and gene therapy.