Biofuel is an attractive substitute for petrodiesel because of its lower environmental footprint. For instance, the polycyclic aromatic hydrocarbons (PAH) emission per fuel energy content is lower for rapeseed methyl ester (RME) than for petrodiesel. The present study assesses genotoxicity by extractable organic matter (EOM) of exhaust particles from combustion of petrodiesel, RME and hydrogenated vegetable oil (HVO) in lung epithelial (A549) cells. Genotoxicity was assessed as DNA strand breaks by the alkaline comet assay. EOM from combustion of petrodiesel and RME generated the same level of DNA strand breaks based on equal concentration of total PAH (i.e. net increases of 0.13 [95% confidence interval (CI): 0.002, 0.259 and 0.12 [95% CI: 0.01, 0.24] lesions per million base pairs, respectively). In comparison, the positive control (etoposide) generated much higher level of DNA strand breaks (i.e. 0.84, 95% CI: 0.72, 0.97) lesions per million base pairs). Relatively low concentrations of EOM from RME and HVO combustion particles (<116 ng/ml total PAH) did not cause DNA strand breaks in A549 cells, whereas benzo[a]pyrene and PAH-rich EOM from petrodiesel combusted using low oxygen inlet concentration were genotoxic. The genotoxicity was attributed to high molecular weight PAH isomers with 5-6 rings. In summary, the results show that EOM from combustion of petrodiesel and RME generate the same level of DNA strand breaks on equal total PAH basis. However, the genotoxic hazard of engine exhaust from on-road vehicles is lower for RME than petrodiesel because of lower PAH emission per fuel energy content.