It is well known that the composition of low-molecular weight glutenin subunits (LMW-GS) in wheat flour is important for end-use quality. However, the contributions of specific LMW-GS to quality have been difficult to assess because of the complexity of LMW-GS within a cultivar as well as the allelic variation between cultivars. Thus, the accurate and reliable determination of LMW-GS alleles in wheat germplasm is very important for breeding efforts. To locate individual LMW-GS corresponding to different alleles encoded by the Glu-A3, Glu-B3 and Glu-D3 loci, we analyzed a set of 15 near isogenic lines (NILs) from Aroona containing unique LMW-GS alleles in the same genetic background. Proteins in glutenin fractions were separated by two-dimensional gel electrophoresis (2-DGE) and the resulting protein patterns were compared to the pattern from Aroona. For most lines, the identifications of protein spots corresponding to LMW-GS alleles were consistent with results using a set of standard wheat cultivars for Glu-3. However, some spots in lines containing the Glu-B3b, Glu-B3g and Glu-D3c alleles differed from the previous study. To confirm their identities, these spots were excised from 2-D gels, digested with chymotrypsin and subjected to tandem mass spectrometry (MS/MS). We also developed a practical and optimized method for RP-HPLC analysis of LMW-GS using a Waters Xbridge BEH, C4 peptide column that results in better resolution than previous studies. The results will be used to identify LMW-GS alleles in germplasm prior to breeding and to screen for desirable LMW-GS alleles in wheat quality improvement.