The Hardness locus on the short arm of chromosome 5D is the main determinant of grain texture in bread wheat. The Pina and Pinb genes are tightly linked at this locus, and the soft kernel texture phenotype results when both genes are present and encode the wild-type puroindoline proteins PINA and PINB. In this study a compensating T5VS 5DL Triticum aestivum-Haynaldia villosa translocation line, NAU415, was characterized by chromosome C-banding, genomic in situ hybridization and molecular markers. Single Kernel Characterization System (SKCS) analysis and scanning electron microscopy indicated that NAU415 had soft endosperm although it lacked the wheat Pina-D1a and Pinb-D1a genes, suggesting the presence of functional Pin gene orthologs on chromosome 5VS. Using a PCR approach, Pina-related (designated Dina) and Pinb-related (Dinb) genes in H. villosa and NAU415 were identified and sequenced. The nucleotide and predicted amino acid sequences showed close similarities to the wild-type puroindolines of T. aestivum cv. Chinese Spring. The tryptophan-rich regions of both Dina and Dinb showed a sequence change from lysine-42 to arginine, a feature that may have an effect on grain texture. The potential of T5VS 5DL translocation line as a source of genes that may be used for modulation of endosperm texture and other valuable traits in wheat breeding is discussed.