Although the field of plant-derived EVs (PDEVs) is experimenting an exponential growth, rigorous characterization complying with MISEV guidelines has not been yet implemented due to the lack of bona fide reference markers. In this work, we have paved the way for the standardization of PDEV markers, providing the most profound proteomic data so far from apoplastic washing fluid-derived EVs, as genuine extracellular vesicles from plant tissue, of two reference plant species: Arabidopsis thaliana (Arath-EVs) and Brassica oleracea (Braol-EVs). Additionally, we have conducted an exhaustive analysis of the EV proteomic data available so far from any plant species, evaluating current potential markers, together with those found in our proteomic analyses. Our results provide evidence supporting the potential use of the following families as PDEV markers: fasciclin-like arabinogalactan proteins (FLAs), patellins, germin-like proteins, aquaporins, heat shock 70 proteins, 14-3-3 like proteins, Rab proteins, and vacuolar-type ATPase complex subunits. Next, we analyzed the presence of orthologues and their degree of conservation throughout the plant kingdom, as well as in human and mouse species. Their degree of conservation was compared with that of current EV markers used in animal-derived EVs: CD63, CD81 and CD9. Among the protein families with potential to be used as PDEV markers, 3 were found to be plant-specific: FLAs, patellins and germin-like proteins. Among these, fasciclin-like arabinogalactan (FLA) proteins, in particular FLA1, FLA2 and FLA8, were recurrently identified in EVs from different plant sources showing great abundance and thus arise as a novel family of plant-specific GPI-anchored PDEV markers. Furthermore, FLAs stand as ideal PDEV markers since they have orthologues across the different plant subdivisions and display a similar phylogenetic distribution in plants to that of currently used markers in animals.