Grain hardness and HMW-GS play a key role in wheat quality. In order to explore and utilize favorable allelic variants of wheat hardness Puroindoline gene and HMW-GS to improve the quality of medium-strong gluten， molecular detection of both genes and quality traits were analyzed in 94 wheat germplasms which mainly include varieties in the Low and Middle Yangtze River Valley Wheat Zone， as well as high quality wheat varieties or landraces in other wheat zones. The results showed that the grain hardness was ranged from 7.21 to 72.91. There were 42 soft wheat accounting for 44.68%， 42 hard wheat accounting for 44.68%， and 10 mixed wheat accounting for 10.64%. Five alleles at the Puroindoline locus were detected， including Pina-D1b/Pinb-D1a，Pina-D1r/Pinb-D1a，Pina-D1s/Pinb-D1a，Pina-D1a/Pinb-D1b and Pina-D1a/Pinb-D1p， with quantity of 8， 3， 1， 29 and 9 respectively. The hardness of different Puroindoline alleles ranked in the order： Pina-D1r/Pinb-D1a＞Pina-D1s/Pinb-D1a＞Pina-D1b/Pinb-D1a＞Pina-D1a/Pinb-D1p＞Pina-D1a/Pinb-D1b. HMW-GS subunits 1 and Null accounted for 53.33% and 45.56% respectively at Glu-A1 locus and one with 1^G330E. 7+8 and 7+9 subunits accounted for 47.78% and 46.67% respectively at Glu-B1 locus. Furthermore， there were three wheat varieties carrying 14+15 subunits， one carrying 7^OE+8^* and one carrying 6+8 at Glu-B1 locus. 2+12 and 5+10 subunits accounted for 61.11% and 38.89% respectively. For SDS sedimentation value， there were no significant difference between 1 and Null subunits at Glu-A1 locus and 7+8 and 7+9 at Glu-B1 locus. The SDS sedimentation value of 5+10 subunits was higher than 2+12 subunits at Glu-D1 locus. A significant positive correlation between hardness and SDS sedimentation value was detected. The SDS sedimentation value was contributed by grain hardness， greater than that of HMW-GS. The genotypes of Puroindoline and HMW-GS and phenotypes of wheat germplasms were analyzed in this study， which provided a reference for the genetic improvement of wheat quality， especially for medium-strong gluten wheat.