High molecular weight glutenin subunits (HMW-GS) in the doubling haploids from stable cultivars and the crossing hybrids between different varieties of common wheat were analyzed to explore the possibility of improving the subunit compositions by anther culture and commercial crossing. Double haploids from five wheat genotypes of Alondra, Orofen, Xinchun9, Verry, and Bainong3217 were obtained by anther culture, and different hybrids were made each other from different wheat lines with different HMW-GS constitutions such as Xingchun9, Jing771, CB037, CS, Ningchun4, Bobwhite, and yangmai12 by crossing. Then, SDS-PAGE was used to analyze the populations of the doubling haploids and the crossing hybrids for HMW-GS combinations. The results indicated that frequent variation of HMW-GS happened in the doubled haploids with a rate up to 61.8% in the test genotypes, among which Alondra and Bainong3217 gave higher variation frequency than other three varieties, and several subunits might be new ones which are not present in the corresponding wild types, but need to be identified further. Comparing with the donor lines, the typical double haploids varied in HMW-GS were not changed in main agronomic characteristics such as plant height, growth period, and grain weight. In most F1 hybrids, the expression of all HMW-GS appeared to be co-dominant, but the expression of one or two HMW-GSs was did found to be suppressed in a few F1 crosses. Cytoplasm of female parents was found to have some effect on the expression of very few subunits in a few crosses. At the same time, 2-3 possible new subunits that did not exist in the parents were observed in the two crosses, Ningchun4/CB037 and Jing771/Ningchun4. By continuous self-crossing and tracing of the new subunits, stable lines expressing the putative new subunits were obtained from the two crosses mentioned above. Variation of HMW-GSs constitution or new HMW-GS can be induced by anther culture and normal cross. Co-dominant of HMW-GS expression did not definitely exist in all F1 crosses. Cytoplasm did effect the expression of HMW-GS in some cases. The present study is theoretical and practical valuable for the improvement of wheat processing quality and the further understanding of the genetic and structural features of HMW-GSs encoding genes.