Sunflower is a globally important oilseed, food, and ornamental crop. This study seeks to investigate the genotoxic effects of tissue culture parameters in sunflower calli tissues belongs to two genotypes obtained via anther culture. Anthers were pretreated with cold for 24 hours at 4 degrees C and heat for 2 days at 35 degrees C in the dark and plated onto media supplemented with different concentrations and combinations of 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, alpha-naphthalene acetic acid and indole-3-acetic acid. Obtaining calli tissues were used to detect the DNA damage levels by Comet assay, evaluating changes on superoxide dismutase and guaiacol peroxidase activities derived from in vitro culture factors. 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 2 mg/L alpha-naphthalene acetic acid from plant growth regulators showed acute genotoxic effect while 0.5 mg/L indole-3-acetic acid and 0.5 mg/L alpha-naphthalene acetic acid showed no genotoxic effect. Total protein content analysis of antioxidant enzymes revealed that although superoxide dismutase activity did not increase, Guaiacol peroxidase (GPOX) activity decreased in comparison to control. The obtained results have indicated that in vitro culture factors apparently lead to genotoxicity and oxidative stress.