Background: Genus Vicia is a member of family Fabaceae and comprises 180 to 210 species. The most important species is faba bean (Vicia faba) which is still one of the most favourable grain legumes over all the world. The genus contains some additional food crops and a number of forage plants and some other weedy strains such as Vicia angustifolia and Vicia cordata. The aim of the present investigation is to elucidate the phylogenetic relationships among four Vicia species, two species (Vicia angustifolia L. ssp. Angustifolia (2n = 12) and Vicia cordata wulfen ex Hoppe (2n = 10)) belong to section Vicia, Vicia dalmatica A. Kern (2n = 12, section Cracca), and Vicia johannis tamamsch (2n = 14, section Faba). Results: Two tools have been applied to identify the genetic relationships among the examined species, double fluorescence in situ hybridization (FISH) has been used to localize the sites of 5S and 45S rDNA, and sodium dodecyl sulfate-poly acrylamide gel electrophoretic (SDS-PAGE) patterns of total seed storage protein fractions. Double FISH experiment has not shown any variation in the loci number, but the positions along the chromosomes were different; both Vicia johannis and Vicia dalmatica exhibited the same interstitial 45S rRNA gene loci, while Vicia angustifolia and Vicia cordata have shown single large stretched 45S rRNA loci almost at the terminal region of the shortest chromosome. It could be concluded from the similarity matrix among the Vicia species as computed according to Jaccard coefficient from the SDS-PAGE, that V. cordata is similar to V. angustifolia and V. dalmatica by a percentage of 73 and 69%, respectively, and the most related species to V. johannis is V. dalmatica (~64%). Conclusion: FISH and SDS-PAGE of the total seed storage proteins together reflected the similar genetic relationship among the studied species as fellows, V. angustifolia is more related to V. cordata then comes V. dalmatica and then V. johannis which is at a distal position from the other species.