Genomic in situ hybridization (GISH) and polyacrylamide gel electrophoresis (PAGE) of wheat seed were used to identify the barley chromosome in offspring of wheat and barley hybrid. A sires of alien addition lines, alien substitution lines and translocation lines were detected by GISH. WBA984 and WBA9812 were alien addition lines, WBS0215 and WBS0264 were alien substitution lines,and WBT02125 and WBT02183 were translocation lines in chromosome terminal. SDS-PAGE analysis and A-PAGE patterns indicated that WBA9812 were 5H alien addition line, WBS0264 was 1 B/5H alien substitution line, and WBT02125 was 1 BL/5HL translocation line.