In this study, wheat high molecular weight glutenin subunits (HMW-GS) 1Bx14 and 1By15 were isolated by preparative SDS-PAGE. They were used as antigen to inject Balb/c mice. Subcutaneous inoculation of the antigen was performed, and intraperitoneal injection was completed 3 days before fusion with myeloma cell (sp2/0) via PEG-2000. The fusion cells were selected by indirect enzyme-linked immuno-sorbent assay (ELISA). The positive hybrid cells were further verified three times by limited dilution of the culture cells. Two hybridoma cell lines were successfully obtained. The classes and subclasses, titers of the two cell lines were identified. Their Mab belong to lgG1 subclass. The titers of the Mab in cultivars superants and ascites fluid were 1:100 and 1:104, respectively.The two Mab have similar specificity patterns in immunblot reaction with wheat HMW-GS after SDS-PAGE. They bound to all HMW-GS of T. aestivum cultivars, but didn’t bind to other storage proteins in seeds of wheat. This result is consisting with the high homology in amino acid sequences between the HMW-GS of wheat. The Mab also bound to in Aegilops squarrosa, T. dicocoides spp. T. durum and Secale cereal (rye), but never did react with seed storage proteins in cereal crops such as barely, maize, oat, rice, millet, foxtail millet, sorghum etc.A wheat endosperm expression cDNA library 16 days after anthesis has been screened with the Mab. Five positive cDNA clones were isolated, purified and sequenced. The sequence data indicated that the inserts belong to HMW-GS genes. This also illustrated the potentiality of the Mab as probe for isolation of HMW-GS coding genes from Triticum and its close relatives.