Technical Abstract: The genome organization of the Hardness locus in the tribe Triticeae constitutes an excellent model for studying the mechanisms of evolution that played a role in the preservation and potential functional innovations of duplicate genes. Here we applied the nonsynonymous-synonymous rate ratio (dN /dS or ') to measure the selective pressures at the paralogous puroindoline-a (Pina), puroindoline-b (Pinb) and Grain softness protein-1 (Gsp-1) genes located at this locus. Puroindolines represent the molecular-genetic basis of grain texture. In addition, the puroindoline gene products have antimicrobial properties with potential role in plant defense. We document the complete coding sequences from the Triticum/Aegilops taxa, rye and barley including the A, D, C, H, M, N, R, S, and U genomes of the Triticeae. Maximum likelihood analyses performed on Bayesian phylogenetic trees showed distinct evolutionary patterns among Pina, Pinb, and Gsp-1. Positive diversifying selection appeared to drive the evolution of at least one of the three genes examined suggesting that adaptive forces have operated at this locus. Results evidenced positive selection (' > 4) at Pina, and detected amino acid residues along the mature PIN-a protein with high probability (> 95%) of having evolved under adaptation. We hypothesized that positive selection at the Pina region is congruent with its role as a plant defense gene.