Adeno-associated viral (AAV) vectors represent an ideal vehicle for human gene transfer. One advantage to the AAV vector system is the availability of multiple naturally occurring serotypes that provide selective tropisms for various target cells. Strategies to enhance the properties of the natural AAV isolates have been developed and can be divided into two approaches, rational design or directed evolution. The rational design approach utilizes knowledge of AAV capsids to make targeted changes to the capsid to alter transduction efficiency or specificity, while the directed evolution approach does not require a priori knowledge of capsid structure and includes random mutagenesis, capsid shuffling, or random peptide insertion. In this study, we describe the generation of novel variants for both AAV2 and AAV5 using a rational design approach and knowledge of AAV receptor binding, surface charge, and AAV capsid protein posttranslational modifications. The novel AAV2 and AAV5 variants demonstrate improved transduction properties in both the mouse retina and cornea. The translational fidelity of the novel AAV2 variant was confirmed in the context of the nonhuman primate (NHP) retina, whereas a NHP tissue explant model was established to allow the rapid assessment of translational fidelity between species for the AAV5 variants. The capsid-modified AAV2 and AAV5 variants described in this study have novel attributes that will add to the efficacy and specificity of their potential use in gene therapy for a range of human ocular diseases.