With 10,700 species distributed in 637 genera, the Rubiaceae family is one of the largest of the angiosperms. Since it was previously evidenced that the fatty acid composition of photosynthetic tissues can be a tool for chemotaxonomic studies, the fatty acid composition of leaves from 107 Rubiaceae species highly representative of the diversity of the family was determined. Principal component analysis allowed a clear-cut separation of Coffeae, Psychotrieae and Rubieae. The occurrence of C16:3 fatty acid, a marker of the prokaryotic plastidial lipid biosynthetic pathway, concerned at least two branches: Theligoneae/Rubieae and Anthospermeae-Anthosperminae which appeared to be in close relationship. Additional experiments were carried out to ensure the correlation between the presence of C16:3 fatty acid and the prokaryotic biosynthetic pathway.