Distinct cell-types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High-throughput analysis of morpho-rheological features has recently been introduced using real-time deformability cytometry (RT-DC) providing new insights into the properties of different cell-types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell-derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell-derived retinal organoids, we characterized the inherent morpho-mechanical properties of mouse rod photoreceptors during development based on RT-DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label-free sorting of photoreceptors. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.