Background: For stable induction of tetraploidy in Fallopia multiflora Haraldson, colchicine was treated to establish the condition of induction and investigated the morphological and cytogenetic traits of the tetraploid plants obtained compared to those of diploid ones. Methods and Results: For the induction of tetraploidy, F. multiflora plants were soaked in aqueous solutions of colchicine at various concentration (0.1, 0.5, and 1.0%). After this, 2% dimethyl sulfoxide (DMSO) was added at room temperature on a shaker set at 150 rpm for periods of 12, 24, and 48 h. The induction rate of tetraploids appeared to be the highest in plants treated with 0.5% colchicine for 24 h. As the colchicine concentration and soaking time increased above these levels, the growing tip of the roots did not develop and they began to rot. When compared to diploid plants, tetraploids differed greatly in various characteristics, including the sizes and shapes of the leaves, fruits, flowers and roots. The induced tetraploid F. multiflora had larger guard cells, and chloroplasts, increased number of chloroplast in the guard cells and decreased stomatal densities. Conclusions: When colchicine induced plants for tetraploid, it can be distinguished from diploids, in various characteristics such as morphological changes as stomatal size, number of chloroplasts per guard cell, number of chromosomes and flow cytometry. Therefore, it proved that these methods are suitable, quick and easy methods for the identification of the ploidy level of F. multiflora.