Therapeutic gene delivery from an oncolytic adenovirus (Ad) is one approach to enhancing the potency of Ad-based virotherapies for cancer. To identify therapeutic transgene insertion sites compatible with the replicating virus, a methodology that broadly scans the viral genome is needed. To address this we modified a transposon (Tn7)-based in vitro transposition system to take advantage of its nonprejudiced scanning ability to identify insertion sites compatible with viral replication. Using this system with a plasmid containing an E3-deleted Ad5, we identified several unique sites for promoter-based expression cassette insertions within the Ad genome. The transposon-based expression cassette is bounded by PmeI restriction endonuclease sites unique to the transposon, making expression cassette substitutions easy to perform. Additional expression cassettes containing different promoters and reporter genes were substituted into two of the newly identified transgene insertion sites. The results suggest that the ease and orientation of expression cassette substitution depend on both the insertion site location and the promoter and gene of the replacement expression cassette. These studies establish the transposon-based system as an efficient approach to scanning the Ad genome and identifying insertion sites compatible with viral replication and represents a powerful tool for the development of armed therapeutic viruses for cancer.