The Trigonella species possess medicinal, nutraceutical and pharmaceutical properties due to the presence of many bioactive compounds. Its therapeutic effects are mostly valuable in medicine, cosmetics and the functional food industry. Correct genetic characterisation of plant material is needed to increase the potential of Trigonella species by breeding and conservation programs. The aim of this study was to develop a reliable marker system to support the morphological and phytochemical analysis in Trigonella taxonomic research, species identification and characterization as well as determination of the interspecific variation within this genus along with relationships between species. For this purpose, flow cytometry and SCoT molecular markers were combined. Flow cytometric analyses revealed that Trigonella species possess very small and small genomes. The range of genome sizes was from 1.10 to 5.76 pg/2C, with most species possessing very small genomes (< 2.8 pg/2C). In seeds of 14 species endopolyploid nuclei were detected. Flow cytometric analysis of genome size enabled quick identification of four out of 20 species, while combined with endopolyploidy detection in seeds, facilitated distinction of the next seven species. ScoT molecular markers helped to identify closely related species with similar genome size and cell cycle activity. Therefore, flow cytometry was proposed as the first-choice method for quick accession screening, while the more detailed genetic classification was obtained using SCoT molecular markers.