Sprouts of diploid red clover ‘Stendes Agrais’ were treated with 0.2% colchicine solution. The ploidy level of plants was detected by flow cytometry. Most of the treated plants were mixoploids and sterile. From progenies of fertile plants, mixoploids were selected that had a proportion of tetraploid cells higher than 60%. In the next generation, stable tetraploid plants were selected, and 49 lines were included in the breeding process.