The root of Polygala tenuifolia Willdenow has been used for the treatment against insomnia, amnesia, depression, palpitations with anxiety, and memory improvement. However, there is no sufficient background information on toxicological evaluation of the root to given an assurance of safety for developing dietary supplements and functional foods. As part of a safety evaluation, the potential genotoxicity of the root extract of P. tenuifolia was evaluated using a standard battery of tests (bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay). In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, the extract did not increase the number of revertant colonies in any tester strain with or without metabolic activation by S9 mix, and did not cause chromosomal aberration in short-period test with the S9 mix or in the continuous (24 h) test. A bone marrow micronucleus test in ICR mice dosed by oral gavage at doses up to 2000 mg/kg/day showed no significant or dose dependent increase in the frequency of micronucleated polychromatic erythrocytes (PCE). These results indicate that ingesting the rot extract P. tenuifolia is not genotoxic at the proper dose.