In this study, we evaluated the storage proteins, macro- and micromorphological characters of seeds from 49 species belonging to 34 genera and seven subfamilies of the Malvaceae s.l. (i.e,  Malvaceae s.s., Bombacaceae, Sterculiaceae, and Tiliaceae) along with one representative of the Elaeocarpaceae as an outgroup. We obtained microphotographs of the specimens using both light and scanning electron microscopes. The seed storage protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We analyzed 73 recorded characters (with 157 characters-states) were analyzed using the upgraded WinClada and TNT cladistic programs. A cladogram was constructed which indicated a clear indication about the systematic position between the studied species within the Malvaceae s.l. The results showed specific synapomorphic diagnostic characters are quite significant in species delimitation within the Malvaceae s.l. viz., including seed shape, apex, base, colour, size, texture, hilum shape, position, seed coat sculpture patterns, and occurrence of strophioly. Our results also support the monophyly of the studied genera in Malvioideae except for Gossypium and Thespesia, while para-polyphyly in the other six subfamilies.