This study aimed to induce autotetraploidy in endemic Onobrychis elata plants by colchicine treatment of seeds or seedlings. Colchicine was applied to O. elata directly on germinated seeds, pre-germinated seeds (root length of 3-8 mm), and apical regions (using cotton) under in vivo conditions. Out of a total of 1,210 colchicine-treated seeds that were evaluated, only 203 survived. There was an inverse relationship between the number of surviving plants and colchicine concentration and exposure time. The highest percentage of tetraploidy in surviving plants (50%) was obtained by applying 0.2% colchicine for 6 hours to pre-germinated seeds. No significant tetraploidy was achieved by colchicine application to seedlings. Flow cytometry observations indicated that DNA content varied between 0.99 and 1.06 pg in diploid plants (controls), while DNA content varied between 2.22 and 2.48 pg in tetraploid plants. It was concluded that tetraploid plants were induced successfully only in seedlings obtained from pre-germinated seeds, with their ploidy level confirmed via flow cytometry analysis.