Twelve AAV serotypes have been described so far in human and nonhuman primate (NHP) populations while surprisingly high diversity of AAV sequences is detected in tissue biopsies. The analysis of these novel AAV sequences has indicated a rapid evolution of the viral genome both by accumulation of mutations and recombination. This chapter describes how this rich resource of naturally evolved sequences is used to derive gene transfer vectors with a wide array of activities depending on the nature of the cap gene used in the packaging system. AAV2-based recombinant genomes have been packaged in dozens of different capsid types, resulting in a wide array of "pseudotyped vectors" that constitute a rich resource for the development of gene therapy clinical trials. We describe a polymerase chain reaction-based molecular rescue method for novel AAV isolation that uses primers designed to recognize the highly conserved regions in known AAV isolates and generate amplicons across the hypervariable regions of novel AAV genomes present in the analyzed sample.