Efficient AAV-mediated gene delivery remains a significant obstacle to effective retinal gene therapies. Here, we apply the process of directed evolution – guided by deep sequencing and followed by direct in vivo secondary selection of high-performing vectors with a GFP-barcoded library – to create AAV viral capsids with new capabilities to deliver genes to the outer retina in large animals. The resulting vectors resulted in efficient targeting of photoreceptors, bipolar cells, and RPE cells in dog retina and substantially increased efficiency of gene delivery to primate outer retina. In addition, direct comparison of gene delivery across animals revealed emergent species specificities in vector performance. These new viral vectors will enable long-term and pan-retinal gene therapies targeting outer retina cell types in large preclinical animal models and establish deep sequencing-guided directed evolution as a powerful approach for developing AAV vectors specialized for a multitude of physical barriers and cellular targets.