Plasmodesmata are cytosolic bridges, lined by the plasma membrane and traversed by endoplasmic reticulum; plasmodesmata connect cells and tissues, and are critical for many aspects of plant biology. While plasmodesmata are notoriously difficult to extract, tissue fractionation and proteomic analyses can yield valuable knowledge of their composition. Here we have generated two novel proteomes to expand tissue and taxonomic representation of plasmodesmata: one from mature Arabidopsis leaves and one from the moss Physcomitrium patens and leveraged these and existing data to perform a comparative analysis to identify evolutionarily conserved protein families that are associated with plasmodesmata. Thus, we identified ß-1,3-glucanases, C2 lipid-binding proteins and tetraspanins as core plasmodesmal components that likely serve as essential structural or functional components. Our approach has not only identified elements of a conserved plasmodesmal proteome, but also demonstrated the added power offered by comparative analysis for recalcitrant samples. Conserved plasmodesmal proteins establish a basis upon which ancient plasmodesmal function can be further investigated to determine the essential roles these structures play in multicellular organism physiology in the green lineages.