Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression in a wide variety of animals, including primates. The rAAV genome is packaged into the virion as single-stranded DNA devoid of any viral genes. A proportion of the single-stranded genomes are converted into transcriptionally active double-stranded DNA (dsDNA) early after nuclear entry by second-strand synthesis mediated by host repair DNA polymerases or/and by annealing of the rAAV (-) and (+) strands. Second-generation, self-complementary vectors are packaged as single-strand hairpins and rapidly assume a dsDNA conformation independent of the action of polymerases. In both cases, linear dsDNA vector genomes circularize and can undergo concatemerization into higher order forms (McCarty et al. Annu Rev Genet 38: 819-845, 2004; Schultz and Chamberlain Mol Ther 16: 1189-1199, 2008; Duan et al. J Virol 72: 8568-8577, 1998). As a result, rAAV vector genomes are maintained mainly as circular monomeric and concatemeric episomal forms in skeletal muscle and liver (Schnepp et al. J Virol 77: 3495-3504, 2003; Penaud-Budloo et al. J Virol 82: 7875-7885, 2008; Nakai et al. J Virol 75: 6969-6976, 2001). Moreover, in nonhuman primate skeletal muscle, it has been shown that rAAV episomes assimilate into chromatin with a typical nucleosomal pattern that presumably is important for persistence and gene expression in quiescent tissues over a period of several years (Penaud-Budloo et al. J Virol 82: 7875-7885, 2008). Conversely, although rAAV is not considered as an integrative vector per se, introduction of exogenous DNA into the nuclear compartment can result in low-level vector assimilation into the host genome. One mechanism appears to involve vector insertion at sites of double-strand DNA breaks using cellular DNA repair enzymes. As rAAV gene transfer technology and applications mature, a better characterization of the genetic fate of the rAAV genome is critical to accurately evaluate the risk/benefit ratio for a particular disease indication. In this chapter, two complementary methods are detailed to enable characterization of rAAV molecular structure in a particular target tissue and estimation of its integration frequency.