The principle of customized endonuclease-triggered genome modification offers unprecedented opportunities for the elucidation of gene functions and the improvement of crop plant performance. The advantage of this technology lies in the possibility of defining the genomic site where a modification is to be made. This chapter provides an overview of the principles of targeted mutagenesis, the state-of-the-art, current limitations, applications of the technology as well as future perspectives on the use of customizable endonucleases in research and breeding devoted to wheat and barley.