Background Highly pathogenic avian influenza (HPAI) poses a tremendous threat to human health and possesses considerable pandemic potential due to the ongoing H5N1 panzootic. HPAI, which includes the influenza A virus (IAV) H5N1 and H7N9 subtypes, has a case fatality rate of 40-50% in humans historically, and an HPAI pandemic could have devastating consequences. The development of effective prophylactic and therapeutic countermeasures for containment of HPAI infections in humans is therefore of paramount importance for pandemic preparedness efforts. Methods To facilitate the development of efficacious HPAI vaccination regimens and the isolation of HPAI-neutralizing monoclonal antibodies (nmAbs) for prophylactic and therapeutic use, we developed an array of novel mRNA-lipid nanoparticles (mRNA-LNPs) and recombinant adenovirus type 5 (rAd5) vectors encoding various HPAI hemagglutinins (HAs), then assessed their immunogenicity in three distinct vaccination regimens in six IAV-naïve Indian rhesus macaques (RMs; Macaca mulatta). Results Binding Abs recognizing the H5N1 and H7N9 HAs were detectable in all six RMs following vaccination. HPAI-neutralizing Abs were detected in RMs vaccinated with full-length HAs, but not in RMs vaccinated with HA stems alone. A heterologous prime-boost-boost regimen comprising mRNA-LNPs and a rAd5 vector encoding the full-length HAs of one H7N9 strain and two different H5N1 strains elicited potent HPAIneutralizing Abs in both animals receiving this regimen, with peak half-maximal inhibitory serum dilution (ID50) values of ~ 1:100,000 against H5N1 and ~ 1:50,000 against H7N9. Our vaccination regimens also elicited cross-reactive Abs recognizing the HAs of seasonal IAV subtypes H1N1 and H3N2, although H1N1 reactivity was superior to H3N2 reactivity in most vaccinees. HA-specific B cells were readily detectable within vaccinee PBMCs using our fluorophore-conjugated HA probes. Conclusion In this study, we demonstrate the immunogenicity of our HA-encoding vaccines and the efficacy of our novel HPAI vaccination regimens in eliciting both HPAI-binding and neutralizing Abs in nonhuman primates. Exceptionally high serum HPAI neutralization activity was observed in two of our animals—the most potent HPAI serum neutralization activity ever recorded in a primate. These data suggest that our vaccination regimen could elicit similar responses in humans. Furthermore, we demonstrate the efficacy of our fluorophore-conjugated HA probes in identifying HA-specific B cells in circulation, which could be used to isolate potent and broadly-reactive HPAI-nmAbs in the future. Collectively, our findings provide insight into vaccine-induced humoral immune responses against HPAI and could facilitate the development of both prophylactic and therapeutic agents for HPAI infections of humans to enhance pandemic preparedness.