This paper evaluates the genotoxicity of vehicular emissions of diesel (B0) and commercial biodiesel (B80) from three different feedstocks (soybean oil, animal fat, and palm oil), based on the emission characterization and the use of Tradescantia bioassays, under controlled conditions. The use of palm biodiesel reduced the toxicological impact of vehicle emissions, which would be the recommended option when incorporating this type of fuel into an automobile fleet. Tradesscantia as biomonitor was sufficiently sensitive and therefore useful to evaluate genotoxicity in complex samples of pollutants, e.g., vehicular emissions.