Abstarct: Plant chromosome manipulation is one of the powerful methods of plant breeding due to its effect on some genetic traits and diversity. Besides, colchicine is a polyploidy inductive compound that has been used successfully to increase ploidy levels in plant species. In order to investigate the effect of polyploidy induction in anise (Pimpinella anisum L.), two different concentrations of colchicine (0.0001, 0.005 and 0.05) were investigated. In this study, the seeds and terminal buds of the five-week-old seedlings were separately treated with the mentioned colchicine concentrations and were repeated for three consecutive days. Besides, molecular, physiological and morphological traits in control (diploid) and induced (auto-tetraploid) plants, which were created by terminal bud treatment, were investigated. The results showed that colchicine at 0.001 had no significant effect on seedlings, while significant effects were observed in 0.005 and 0.05 concentrations. Larger stomatal size, lower stomatal density and darker leaf color were observed in seedlings treated with concentrations higher than 0.005 colchicine concentrations. The content of DNA, RNA and total protein in these seedlings also increased. In general, the findings of this study indicated that colchicine can change the ploidy level of anise at 0.005 and 0.05 and increase the leaf size and pigments associated with photosynthesis and thus produce stronger plants. The results of this study are the first report on the production of auto--tetraploid seedlings in Anise, and these findings could be useful in future plant breeding programs related to this plant species.