Genotoxicity detection is an important part of food safety assessment because many food contaminants are genotoxic. Through the regulation of gene expression, causing DNA damage, inducing gene mutation, and influencing DNA methylation, such contaminants pose a severe threat to the health of human and animals and can even induce cancer. The development of PCR technology makes it possible and easier to detect genotoxicity at the molecular level. Of the various genotoxic pathways, gene mutation, DNA methylation, and miRNA regulation are issues of particular concern. This chapter therefore focuses on the technologies for the detection of gene mutation, DNA methylation, and miRNA and reviews the conventional methods and developments in these fields. Direct DNA sequencing, PCR-SSCP, PCR-RFLP, ASO-PCR, biosensors, SNP detection, and other methods are discussed in the context of gene mutation detection. With regard to DNA methylation detection, techniques for both genome-wide methylation and gene-specific methylation are reviewed. In the discussion of miRNA detection, we introduce two of the most easily accessible and widely used methods for this purpose, namely, poly(A)-tailing PCR and stem-loop PCR, as well as innovative techniques such as biosensors. Finally, the prospects under development in these fields are discussed.