Large double-stranded DNA (dsDNA) viruses such as adenovirus (Ad), herpesvirus, poxvirus and their recombinant counterparts were well explored in basic virology and biomedical research. Especially for the development of novel vaccines and gene therapies, Ad gained special attention and represents the most widely explored vector worldwide. Although ~70 types of human Ad and numerous nonhuman Ad (>200) have been identified so far a system for efficient Ad genome cloning and manipulation was lacking and therefore the majority of recombinant adenoviral vectors (AdVs) are based on a small fraction of Ad types. Here we report the generation and characterization of an engineered human adenoviral-library allowing exploration and system studies of the natural Ad diversity. Towards that end we first established that adenoviral genomes can be cloned and tagged in a high-throughput manner utilizing advanced homologous recombination techniques. Wild type Ads from clinical isolates including around half of the currently known adenovirus types that represent all seven human adenovirus species were propagated and direct high-throughput cloning (HTC) applied. The integrity of cloned Ad genomes was confirmed by DNA restriction enzyme pattern and virus reconstitution was conducted using optimized conditions. Most importantly next-generation sequencing (NGS) and phylogenetic analysis was performed. As a further step, half of all cloned adenoviruses representing each species were tagged with a 2A peptide-mediated multicistronic expression cassette providing a Turbo Green fluorescent protein as in vitro marker, a NanoLuc luciferase for in vivo studies and kanamycin/neomycin as selection marker. For HTC of tagged viruses the reporter cassette was inserted into the adenovirus E3 region using different orientations because the orientation of the transgene was essential for reconstitution efficiencies. After successful reconstitution these double reporters-labeled AdVs were evaluated in cultured cell lines and mouse models. In vitro characterization revealed distinct tropisms for tested viruses. Among the currently evaluated cell lines, specie B viruses demonstrated high transduction efficiencies in epithelial (Hela- and A549 cells) and endothelial cells, while Ad5 still displayed highest transduction rates in other human and murine cell types (hepatocytes, lymphocytes, neuroblastoma cells and myoblasts). For the further characterization of unknown receptor usage, we injected recombinant viruses into DSG2 or CD46 transgenic mice for in vivo bio-distribution analyses on genome level by quantitative PCR and on protein level by immunohistology analyses. We anticipate that our engineered adenoviral-library will provide a spacious novel view to the adenovirus field. As a broader perspective it will bring AdV and also other large dsDNA viruses from mono- to multi-types and enables broader applications in molecular medicine including gene therapy and vaccination studies, as well as basic virology.