Locating of important agronomic genes onto chromosome is helpful for efficient development of new wheat varieties. Wheat chromosome substitution lines between two varieties have been widely used for locating genes because of their distinctive advantages in genetic analysis, compared with the aneuploid genetic materials. Apart from the substituted chromosome, the other chromosomes between the substitution lines and their recipient parent should be identical, which eases the gene locating practice. In this study, a set of chromosome substitution lines with cv. Wichita(WI) as the recipient parent and cv. Cheyenne(CNN) as the donor parent were studied for the composition of high molecular weight glutenin subunits(HMW-GS) as well as a range of agronomic important traits. Results revealed that the substitution lines of WI(CNN5D), WI(CNN6A) and WI(CNN7B) had higher plant heights than the two parents of WI and CNN, and WI(CNN3D) had later maturity than the parents. By sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) analysis, a substitution line WI(CNN5B) was found to contain different HMW-GS patterns from its two parents, in which 1By9 was replaced by 1By8 on chromosome 1BL. Simple sequence repeat(SSR) analysis confirmed that the variation on 1BL in WI(CNN5B) was originated from Chinese Spring(CS). It is concluded that chromosomal fragments from bridge material and donor parent were quite often retained in intracultivaral chromosome substitution lines except the substituting chromosomes. Locating of important agronomic genes onto chromosome is helpful for efficient development of new wheat varieties. Wheat chromosome substitution lines between two varieties have been widely used for locating genes because of their distinctive advantages in genetic analysis, compared with the aneuploid genetic materials. substituted chromosome, the other chromosomes between the substitution lines and their recipient parent should be identical, which eases the gene locating practice. In this study, a set of chromosome substitution lines with cv. Wichita (WI) as the recipient parent and cv. Cheyenne (CNN) as the donor parent were studied for the composition of high molecular weight glutenin subunits (HMW-GS) as well as a range of agronomic important traits. Results that the substitution parent of WI (CNN5D), WI (CNN6A) and WI (CNN7B) had higher plant heights than the two parents of WI and CNN, and WI (CNN3D) had later maturity than the parents. By sodium dodecyl sulfate po lyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, a substitution line WI (CNN5B) was found to contain different HMW-GS patterns from its Simple sequence repeat (SSR) analysis confirmed that the variation on 1BL in WI (CNN5B) was originated from Chinese Spring (CS). It is concluded that chromosomal fragments from bridge material and donor parent were quite often retained in intracultivaral chromosome substitution lines except the substituting chromosomes.