Accordingly, we have developed a novel, rapid, high-throughput platform to identify regions of the capsid of any AAV variant that are involved in a specific viral function. Specifically, we have generated highly diverse AAV libraries based on AAV5 and AAV6 through random mutagenesis of the cap gene and selected for mutants that are defective in specific steps of viral infection. For example, to map key residues involved in receptor binding, we selected the libraries for variants with decreased cell binding in iterative rounds of binding to CHO cells. Characterization of clones from the selected libraries yielded >15 novel variants of both AAV5 and AAV6 that exhibit decreased affinity for CHO cells. Interesting, a majority of these residues cluster to distinct regions of the capsid for AAV5 as compared to AAV6. Furthermore, several of these variants demonstrate altered gene delivery efficiencies to mutant CHO cell lines lacking sialic acid or HSPG, suggesting some of these residues are necessary for viral binding to sialic acid and other cellular receptors. This work demonstrates that our novel forward genetics platform is an efficient and effective approach to map functional regions of the AAV capsid and potentially other proteins, further our knowledge of basic AAV biology, and enhance efforts to engineer viruses and other proteins with customized properties.