Toluene is a widely used industrial organic solvent and is ubiquitous in our environment. The neurobehavioral and neurotoxic effects of toluene are well recognized; however, its genotoxicity is still under discussion. Toluene biotransformation leads to the generation of reactive oxygen species that cause oxidative stress and DNA damages. Individuals with different immunogenetic backgrounds have different sensitivities to toxic chemical exposure. Previous studies have suggested that allergic stimulation may influence the threshold for toluene sensitivity due to the modulation of neurotrophin-related genes. Therefore, we aimed to investigate toluene-induced genotoxicity in different brain regions following acute and chronic exposure in vivo and to further examine whether allergic stimulation may influence the sensitivity to toluene-induced genotoxicity. In this present study, we found that exposure of toluene induced oxidative DNA damages resulting in genotoxicity in different brain regions including cortex, cerebellum, and hippocampus using comet assay. Higher genotoxicity induced by toluene was observed in the hippocampus of control mice compared to OVA-immunized mice. These results provide evidence that toluene-induced genotoxicity may contribute to its neurotoxicity in different immunogenetic individuals.