One of the major limitations to the success of non-viral gene therapy has been the low gene transfer efficiency and short transgene expression. Chitosan has shown great promise as a non-viral carrier due to its biocompatibility, biodegradability and low cost, but limited by its low transfection efficiency. Our goal is to develop chitosan-based non-viral vectors optimized for retinal gene therapy and continued gene expression. The gene transfer efficiency and biocompatibility of chitosan-pDNA polyplexes was evaluated both in vitro on retinal pigment epithelial (ARPE-19) cells and human embryonic kidney (HEK293T) cells, and in vivo in the mouse retina. Our results show that chitosan polyplexes have size and surface charge consistent with gene delivery. When considering off the shelf applications, vector stability is crucial, and our vectors are stable both in storage (4 °C) and physiological conditions (37 °C), and remain stable after several freeze-thaw cycles. Despite moderate in vitro efficiency, in vivo sub-retinal administration shows sustained transgene expression in RPE cells at least 6 months post-injection. These results show that chitosan-based vectors can successfully transfer genes to the retina and provide persistent transgene expression.