Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.) that has main effect on milling yield, particle size, and water absorption of wheat flour. Kernel hardness is mainly controlled by Ha locus on the short arm of chromosome 5D in bread wheat. In this study, we have employed a multifaceted approach to investigate aspects of grain hardness in bread wheat cultivars with different origins. We conducted mechanical experiments by compression testing (CT) and near infrared reflectance (NIR). We used STS and SNPs primers by switch temperature PCR for identification of allelic variation in puroindoline genes for Pina-D1b, Pina-D1a, Pinb-D1b, Pinb-D1c, Pinb-D1d and Pinb-D1a in the germplasms. The results indicated that more than half of the cultivars were categorized as hard grains. Other cultivars showed grain softness. In general, the most frequent alleles were Pina-D1b and Pinb-D1b and after that Pinb-D1d allele have the most frequency in cultivars. Suprisingly Pinb-D1c allele was found in none of cultivars. Most of germplasms that showed hardness belong to warm and humid or warm and dry conditions. Coefficient correlation between CT and NIR, molecular results (genetically soft or hard) with NIR and CT (α<0.0001), were r =0.74, 0.87 and 0.79, respectively. This study validated a series of recent STS-PCR and SNPs markers were employed for grain hardness. Our knowledge about the genetic and mechanical basis of kernel hardness could provide useful information for breeders in breeding programs of bread wheat.