Puroindoline b-2 is related to wheat grain hardness and yield-related traits. In this study, 81 wheat cultivars or advanced lines from four different countries were used to identify allelic variation of Puroindoline b-2 by PCR amplification with specific primers and DNA sequencing, and relative expression levels of different Puroindolne b-B2 alleles were examined in grain, leaf and root of selected wheat cultivars by real-time quantitative PCR (RT-PCR). The results showed that all bread wheat cultivars surveyed contained Pinb-D2v1 and Pinb-A2v4 variants in their genome D and A, respectively. However, Pinb-B2v3 variant was identified on the B genome of 74 wheat cultivars and advanced lines, and Pinb-B2v2 variant was found in the remaining 7 cultivars and advanced lines. Therefore, Pinb-B2v3 is the most prevalent variant in Pinb-2 locus with percentage of 91.4℅, while Pinb-B2v2 was only 8.6℅. RT-PCR result indicated that the expression level of Pinb-B2v3 was significantly higher than that of Pinb-B2v2 in wheat grain. Furthermore, the expression level of Pinb-B2v3b variant is significantly higher than those of Pinb-B2v3a and Pinb-B2v3c variants that have no significant difference even though Pinb-B2v3c is slightly higher than that Pinb-B2v3a. In addition, the expression level of Pinb-B2 in leave of wheat was significantly higher than that in roots. This study could provide a better understanding of molecular and genetics basis of Pinb-2 gene.