Gene replacement therapy for retinal degeneration has seen unprecedented results in the last decade which culminated in several successful phase I/II clinical trials for Leber Congenital Amaurosis (LCA), Choroideremia and Age-related Macular Degeneration (AMD) using replication-deficient adeno-associated viral vectors (AAV). AAV vectors have shown extremely high affinity to retinal cells and innumerous pre-clinical studies have demonstrated a consistent success rate for visual rescue and gene delivery in the retina of several animal models of inherited retinal degeneration, validating the use of AAV vectors as a safe and effective gene therapy tool to treat retinal disorders. Despite the highly AAV-permissive profile of the retina, different AAV serotypes offer varying levels of transduction and cell specificity upon delivery to the subretinal space which has created challenges for improving clinical trial design for retinal dystrophies. RPE and rods, and to a lesser extent, cone photoreceptors can be efficiently targeted with AAV. However, other retinal cell types have remained a more challenging target. In an effort to increase transduction efficiency and specificity while improving our understanding of the structure-function relationship of AAVs, our group has recently reconstructed the evolutionary lineage of most of the AAV serotypes under clinical testing using in silico phylogenetic and statistical modeling. We used phylogenetic and bioinformatic methods to infer the ancestral state of AAV and most likely AAV capsid sequences libraries were synthesized and members were evaluated for viral assembly and DNA packaging. Lead candidates were selected and, following subretinal injection into C57Bl/6 mice and non-human primate, retinal tropism was characterized by (a) in vivo transduction by reporter gene expression from fundus imaging, (b) molecular analyses of expression levels, (c) onset of expression and (d) histological profile of targeted retinal cell types. In vivo retinal transduction in mice demonstrated a high level of RPE and photoreceptor tropism that is quantitatively higher than AAV8. In addition, onset of expression of Anc80 was visible as early as day 1 post injection and accelerated compared to AAV8. Several variants of Anc80 were evaluated for inner retinal tropism with evidence for improved transduction of cone photoreceptors, Muller glia, ganglion cells, and other inner INL neurons. Preliminary studies in non-human primates also indicate an accelerated onset of expression compared to AAV9 and AAV5. This study present an in depth evaluation of the efficiency and tropism of three novel ancestral AAV variants for broad retinal gene therapy applications. The variants tested here revealed potent gene therapy vehicles with broad and unique properties that could provide novel alternatives from current vectors.