Seed storage proteins have been used for studying biochemical genetics and end-use quality aspects. We conducted enzyme-linked immunosorbent assay (ELISA) and one-dimensional SDS-PAGE (1D SDS-PAGE) to evaluate different cereal crop species and Korean wheat lines for rye secalin proteins. The antisecalin antibody showed consistent specificity for rye secalin with little cross-reactivity to gliadins. Immunological cross-reactivities measured by the ELISA technique using competition assay showed significant differences of absorbance among rye, triticale, wheat-rye translocated wheat and non-translocated wheat. The absorbance values were lowest in rye followed by triticale, translocated wheat and non-translocated wheat. The ELISA for discrimination of wheat-rye translocation on the basis of antigen-antibody reactivity showed that none of the Korean wheat lines possessed 1RS and secalin proteins. The competitive ELISA experiment demonstrated specific determination for secalin that was originated from rye chromosomal parts. The result of 1D SDS-PAGE for identifying rye secalin subunits showed all three rye specific secalin protein subunits (75 KDa, 45 KDa, and 40 KDa) for rye and triticale, and 1RS specific secalins (45 KDa and 40 KDa) for 1AL/1RS and 1BL/1RS translocated wheats. All Korean wheats were lacking 1RS of rye chromosome and secalin.