Technical Abstract: Cloning and phylogenetic analysis of puroindoline b-2 variants in common wheat (Triticum aestivum L.) and its relatives would advance the understanding of the genetic diversity and evolution of puroindoline b-2 gene in common wheat and its related species. In the present study, common wheat (AABBDD) and five related species, including T. urartu (AuAu), T. boeoticum (AmAm), Aegilops speltoides (SS), Ae. tauschii (DD), and T. dicoccum (AABB) were sampled for the presence of novel alleles at Pinb2v-A1, Pinb2v-B1/Pinb2v-S1 and Pinb2v-D1 loci corresponding to common wheat puroindoline b-2 variants. Ten new alleles were identified at these loci, designated Pinb2v-A1a through Pinb2v-A1d, Pinb2v-S1a through Pinb2v-S1e, and Pinb2v-D1a. Alignment of puroindoline variants or alleles from common wheat and its relatives indicated that all alleles in diploid wheats are attributed to single nucleotide substitution when compared with Pinb-2v genes in polyploids. Deduced amino acid sequences showed that all four alleles at Pinb2v-A1 locus and four alleles (Pinb2v-S1a, Pinb2v-S1b, Pinb2v-S1c and Pinb2v-S1e) at the Pinb2v-S1 locus could not be normally translated due to the presence of premature stop codons, whereas Pinb2v-D1a at the Pinb2v-D1 locus and Pinb2v-S1d at the Pinb2v-S1 locus could be normally translated, possibly suggesting that the Pinb2v gene in Ae. tauschii was more highly conserved than those in T. urartu and Ae. speltoides. Meanwhile, puroindoline b-2 variant could be normally translated in all of the durum and common wheat cultivars. None of the puroindoline b-2 alleles previously identified in durum and common wheat were found in the diploid genome donors examined here, even though a greater diversity of alleles were found in diploid wheat compared to polyploid wheat. These results likely reflect the evolutionary history of tetraploid and hexaploid wheats, although it may be that puroindoline b-2 variant alleles have been selected for stability and functionality in common and durum wheat. This study provides a survey of puroindoline b-2 variants in common wheat and its relatives, and provides useful information for understanding the genetic diversity of puroindoline-like genes and their duplication events in wheat.