Introduction: Clover has attracted considerable interest not only as a valuable livestock forage plant, but also as an important source of isoflavones. The current study was aimed to assess the variation of concentration of three isoflavones in clover species grown under a cool temperate climate environment in Lithuania. Materials and Methods: Isoflavone contents were quantified in the plant parts of 21 accessions belonging to five perennial species of genus Trifolium (T. pratense, T. repens, T. medium, T. rubens, and T. pannonicum). Daidzein, formononetin, and genistein concentrations were quantified in separate plant parts by reversed-phase high-performance liquid chromatography. The analyte extraction was performed from dried and ground leaves, stems, flowers, and roots. The procedure included acid hydrolysis of isoflavone glycosides to aglycones. Results: According to the averaged sum of the three isoflavones quantified in leaves-stems-flowers, the five clover species ranked as follows: T. medium (7.54-3.62-2.31 mg/g) >T. pratense> T. rubens> T. pannonicum> T. repens (0.191-0.204-0.171 mg/g). The contribution of individual compound to the total isoflavone content depended on the species, accession, and plant part. The major part of the isoflavones is concentrated in leaves or stems; however, there is a great variation also. Conclusion: There exists a large variation in the total as well as in individual concentration of isoflavones among the clover species and plant parts and within species. With regard to isoflavone concentration and variability within species, some accessions of T. medium and T. pratense can be considered a highly promising source of phytoestrogens.