Recombinant adeno-associated virus (AAV) is a promising gene therapy vector. To make progress in this direction, the relationship between the characteristics of the genomic cargo and the capsid stability must be understood in detail. The goal of this study is to determine the role of the packaged vector genome in the response of AAV particles to mechanical compression and adhesion to a substrate. Specifically, we used atomic force microscopy to compare the mechanical properties of empty AAV serotype 2 (AAV2) capsids and AAV2 vectors packaging single-stranded DNA or self-complementary DNA. We found that all species underwent partial deformation upon adsorption from buffer on an atomically flat graphite surface. Upon adsorption, a preferred orientation toward the twofold symmetry axis on the capsid, relative to the substrate, was observed. The magnitude of the bias depended on the cargo type, indicating that the interfacial properties may be influenced by cargo. All particles showed a significant relative strain before rupture. Different from interfacial interactions, which were clearly cargo-dependent, the elastic response to directional stress was largely dominated by the capsid properties. Nevertheless, small differences between particles laden with different cargo were measurable; scAAV vectors were the most resilient to external compression. We also show how elastic constant and rupture force data sets can be analyzed according a multivariate conditional probability approach to determine the genome content on the basis of a database of mechanical properties acquired from nanoindentation assays. Implications for understanding how recombinant AAV capsid-genome interactions can affect vector stability and effectiveness of gene therapy applications are discussed.