Gap junctions are widely prevalent in the nervous system and play many pivotal roles including synchronising and collecting of electrical signals. In vertebrates, gap junctions consist of two adjacent hemichannels which in turn are made up of six connexins (Cx). Connexins and their gap junctions emerge long before chemical synapses during neural development, allowing communication between developing cells, and are thought to be involved in establishing distinct neural circuits. It is unclear how much Cx45 and Cx36 are involved in retinal circuit creation in mouse retinas and retinal organoids generated from human induced pluripotent stem cells (ROs) and whether developmental parallels can be found in both systems. Here, we quantified the developmental expression patterns of Cx45 and Cx36 in postnatal day 8-16 mice and compared against ROs differentiated to days 40, 90, 150 and 200.For this, we developed a custom software that automatically analyses multiple anatomical parameters such as size, overlap and near-by location of all channels within a confocal microscopy stack. Our results show that numerous soma-somatic Cx45 and Cx36 gap junctions are existing in ROs, and both connexins were localised on and near synaptic terminals that were labelled with synaptophysin and vesicular glutamate transporter 1 in both tissues. Cx45 and Cx36 expressions in both plexiform layers of the mouse retina increased till eye opening and dropped slightly afterwards. Cx45 expression patterns in ROs showed increasing densities at later differential stages, whereas Cx36 displayed less pronounced expression than in mice. Heterotypic Cx45/Cx36 gap junctions, which can be found as part of the rod pathway between ON cone bipolar cells and AII amacrine cells, were similarly expressed in mice and ROs. The percentage of Cx45/Cx36 gap junctions is higher before the critical event of eye opening in the mouse while in ROs it steadily increased. In addition, our multielectrode array recordings from ROs revealed gap-junction-coupled RGCs, consistent with similar findings from both connexins between retinal ganglion cells (RGCs) in mice. In conclusion, mice and ROs have very comparable Cx45 and Cx36 expression patterns. We show for the first time that ROs have heterotypic Cx45/Cx36 gap junctions and functional gap junctions between RGCs in ROs. These findings imply that both connexins play a key role during development in the mouse retina and ROs.