Pemetrexed is a chemotherapeutic drug, approved for the treatment of mesothelioma and non-small cell lung cancer and variety of neoplasm. Chemotherapeutic drugs don't target only cancer cells in treatment. During treatment these drugs encounter with normal cells as well. Therefore, in this in vitro study, it was aimed to investigate the whether genotoxic effect or not of Pemetrexed on normal leukocytes. In vitro alkaline comet assay was used in this study. Leukocytes from human peripheral blood samples from two volunteer donors were used in the study. In the present study, leukocytes were treated with four different concentrations (25, 50, 75, and 100 mu g/ml) of the drug for 1 hour. At concentration of 75 mu g/ml and 100 mu g/ml, there were statistically significant increases in DNA damage compared to the negative control (p<0.05, and p<0.01 respectively). The obtained result shows that Pemetrexed may have genotoxic potential on normal leucocytes.