Wheat is one of the most widely grown crops in the world. One of the traits that defines wheat quality is grain hardness, which is determined by puroindolines (PINA and PINB) proteins encoded with Pina-D1 and Pinb-D1 genes. In this study, the diversity of Pina-D1 and Pinb-D1 was evaluated in a collection of 271 Iranian common wheat (Triticum aestivum L. ssp. aestivum) landraces, whose kernels had previously been classified as hard or semi-hard based on PSI analysis. Three alleles previously described as associated with hard grain were detected in the collection: Pinb-D1b in 11 accessions, Pinb-d1ab in 175 accessions, and Pinb-d1p in 80 accessions. In addition, a novel allele tentatively named Pinb-d1ak was detected in Pinb-D1 and was characterized by a change at position 140 of the deduced protein (cysteine/tyrosine). On average, the accessions with this allele showed a lower PSI value than the accessions with other Pin allele. This means that this novel allele may be associated with harder grains than other Pin alleles and could be used by breeding programs targeting different grain hardness levels. This study highlights the importance of conserving and characterizing wheat genetic resources that could be used as sources of genetic variability in breeding programs.