Tetraploid plants of ruzigrass (Brachiaria ruziziensis) have been obtained by colchicine treatment of in vitro multiple-shoot clumps and germinated seedlings. Multiple-shoot clumps were induced and maintained on MS basal medium with 0.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 mg L−1 6-benzylaminopurine (BAP) according to a previous report. The colchicine treatment consisted of culturing multiple-shoot clumps on MS basal medium containing 0.0125–0.1% colchicine for 12 and 48 h. Surviving multiple-shoot clumps were regenerated on MS basal medium containing 2.0 mg L−1 BAP. The ploidy level of plants after colchicine treatment was determined by flow cytometry. Eight tetraploid plants were obtained from multiple-shoot clumps treated with 0.1% colchicine for 12 h. This treatment was identified as the optimum treatment and resulted in the highest frequency (28.6%) of tetraploid plants among the treatments tested on multiple-shoot clumps. Five tetraploid plants (31.3%) were obtained when seedlings were treated with 0.1% colchicine for 3 h. Although higher concentrations of colchicine and longer durations of colchicine treatment reduced the survival rate of the explants, such treatments increased the frequency of tetraploid plants. A total of 23 tetraploid plants were obtained from 900 different colchicine-treated multiple-shoot clumps, and a total of five tetraploid plants were obtained from 300 colchicine-treated seedlings. Cytological analysis affirmed the results of flow cytometry. Significant differences in leaf blade stomata were observed between diploid and tetraploid ruzigrass.