During the SARS-CoV-2 pandemic different vaccination approaches have been pursued, one of which based on the use of adenovirus (Ad) vectors for vaccine antigen expression. Next to very good immunization properties, additional advantages of Ad vectors included scalability during production, low production costs and stability of the vaccine. The COVID-19 vaccines developed by Oxford University/AstraZeneca and Johnson & Johnson were based on adenoviruses of chimpanzee (Y25) or human (hAdV-26) origin, respectively, both expressing the SARS-CoV-2 spike (S) protein as antigen. Since a previous exposure to one Ad type during prime vaccination will negatively impact on the immune response during boost vaccination with the same Ad type, we wish to expand the choice of Ad vector types that could be used in prime-boost vaccine regimen. Therefore, we characterized twelve potential alternative human adenovirus (hAdV) types from species B and D in different in vitro assays, including (i) production yield, (ii) target cell binding, and (iii) seroprevalence. hAdV-11, -34, -37, -43, -48 and -50 were identified to be producible to high titers, demonstrated good target cell binding and had a low seroprevalence. The full-length genomes of this sub-selection were introduced into bacterial artificial chromosomes and the E1 genes were replaced by an eGFP-firefly luciferase reporter gene to yield replication-deficient Ad vectors. After further characterization and selection, the E3 genes will be deleted to increase vector stability, DNA transport capacity and vector immunogenicity. Additionally, the Spike-protein will be introduced as the vaccine antigen and vaccination studies will be performed in mice.