The wheat-rye translocation lines have been agriculturally developed for the resistance to the biotypes of Hessian fly as a major insect pest of wheat. In order to compare the proteomic profiles between ‘Coker797’ (non-2RL), ‘Hamlet’ (2RL), and near-isogenic line (NIL) carrying 2RL, we evaluated the protein extraction and preparation methods for two-dimensional gel electrophoresis approach. The tissues such as leaves, stems, and roots from three wheat-rye lines were extracted by following trichloroacetic acid (TCA)/acetone precipitation. In a preliminary proteome analysis, a commonly expressed protein in Hamlet and NIL strain was identified as methionine synthase annotated in Hordeum vulgare subsp. The present study will provide the experimental guideline for the proteomic study of other useful crop plant tissues.