Aims: The feasibility of Escherichia coli DJ 702 lacZ mutagenicity assay to detect genotoxicity of benzidine and its derivatives was evaluated. Methods and Results: DJ 702 strain was grown overnight at 30°C in Luria-Bertani (LB) medium containing some components, such as chloramphenicol, ampicillin, δ-aminolevulinic acid, isopropyl-β-d-thiogalactoside, and trace element mix. The mixtures of a bacterial culture and tested chemical at indicated doses were incubated at 30°C for 30 min. Subsequently, 2 ml of molten top agar was added and the resulting mixtures were immediately poured onto a minimal lactose (ML) plate. Plates were incubated at 30°C for 48 h. The number of colonies was determined by visual scoring. In this study, results showed that all the tested chemicals were mutagenic to DJ 702 strain. Conclusions: E. coli lac mutagenicity assay using DJ 702 strain can detect the mutagenicity of benzidine and its derivatives. Significance and Impact of the Study: We detected the mutagenicity of benzidine and its derivatives in E. coli lac mutagenicity assay using DJ 702, indicating that this assay may be used to detect benzidine and its derivatives in a powerful, sensitive, and convenient mutagenesis assay.