The study of polymorphism of glutenin makes it possible to identify and isolate desirable genotypes with higher grain quality. In the last few years, only a part of the genetic diversity among the modern and popular wheat germplasm and varieties based on the polymorphism of glutenin subunits are captured. To address this 107 wheat varieties released across different agricultural zones in India, were used to investigate HMW-GS and LMW-GS allele polymorphism, gene diversity and genetic variation in the Glu-1 and Glu-3 loci. Among the different HMW-GS, the highest genetic variation was observed at the Glu-D1 locus with both Glu-D1a and Glu-D1d possessing genetic variation of 0.490, 0.484 respectively. The highest genetic variation at the Glu-A3 locus was observed at the Glu-A3c and GluA3b possessing a genetic variation of 0.463, 0.411 respectively. This was followed by the Glu-B3j having a genetic variation of 0.386 at the Glu-B3 locus. Over 20 years a remarkable increase in the Glu-D1d allele is observed in the newly released varieties in India. Among all the zones, Glu-A1-null is the least frequent allele at the Glu-1 locus, however, it is present as the predominant allele in the NHZ of India. This study elucidates the relationships of these HMW and LMW allelic frequencies and genetic variation with their geographical distribution over the two different periods. This study provides reference data that can be used to assist the breeding, quality evaluation and development of good-quality wheat varieties.