Thallium is a heavy metal whose concentration in the environment has been continuously increasing by industrial activities such as metal ore processing, coal combustion in power plants or cement production. Thereby thallium and its compounds become biologically available and are accumulating in living organisms, plants in particular. The objective of this research was to determine genotoxicity of thallium(I) acetate in tobacco Nicotiana tabacum L. var Xanthi by comet assay, which as a non-specific genotoxicity biomarker quickly and simply shows changes in the DNA molecule, and therefore has a wide-range application in ecotoxicological research and biomonitoring. In order to verify the comet assay sensitivity, hydrogen peroxide was used as a model genotoxicant, in vitro conditions showing a direct genotoxic effect on tobacco cell nuclei. Tobacco plants were in vivo exposed to different concentrations of thallium(I) acetate for three days. After that period thallium(I) acetate caused mild damage to DNA molecules of tobacco leaves and greater damage to the root. DNA damages grew linearly as the concentration of thallium increased. An acellular comet assay was performed in vitro on tobacco leaf nuclei to determine possible mechanisms of thallium effects on DNA. The results were similar to those obtained by a cellular comet assay on tobacco root cells while the DNA damage to the leaf was lower but statistically different from the control sample.