ABSTRACT. The manipulation of ploidy is a helpful tool in improving crops and grasses. Trifolium polymorphum (Poir.) (Leguminosae) is a native perennial stoloniferous winter species that combines different types of reproductive systems. This biological advantage makes T. polymorphum a remarkable issue of research. T. polymorphum belongs to the Campos region and has evolved into adapted ecotypes through long exposure to environmental stresses, grazing and by competing with the native C3 and C4 grasses which have dominated the Campos vegetation over a long period of time. Eurasiatic Trifolium species lack persistence in the temperate to subtropical environment found in the Campos region and have to rely on their reseeding capacity to persist. T. polymorphum is currently being studied for its ability to adapt to Uruguayan conditions. Despite this significant advantage in survival, the major constraint of T. polymorphum is a poor biomass production and low seedling vigor that represents a limitation for its use as a forage legume. The objective of this work was to evaluate different in vitro methods to induce polyploidy. We evaluated two antimitotic agents in different concentrations and time exposures:colchicine and oryzalin in different kinds of explant, and the effect of colchicine added in the culture medium. Colchicine was more effective in polyploidy induction. In seedlings 0.3% of colchicine for 24 h achieved 11% of tetraploids, for in vitro explants 0.2% of colchicine reached to 13% of chromosome duplication, and 16% when colchicine (0.015%) was added in the culture medium. The numbers of chloroplasts in stomatal guard cells was used as a quick assessment of the ploidy level and then confirmed by flow cytometry. @ISHS, International Society for Horticultural Science.