Wheat related diploid species Ae. longissima (2n=2x=14, SlSl) has extensive storage protein variations that may provide useful gene resources for wheat quality improvement. In this work, five novel 1Sl-encoded x-type high molecular glutenin subunits (HMW-GS) were identified and designated as 1Slx-123, 1Slx-129, 1Slx1-136, 1Slx2-136 and 1Slx2.2, respectively. Their complete open reading frames ... [Show full abstract] (ORFs) were cloned and sequenced by AS-PCR, which contained 2874 bp (956 aa) for 1Slx-123, 2946 bp (979 aa) for 1Ssx-129, 2901 bp (965 aa) for 1Slx1-136, 2982bp (991 aa) for 1Slx2-136 and 2928 bp (974 aa) for 1Slx2.2. Molecular characterization demonstrated that five unusual subunits had greater repetitive domains resulted from a larger fragment insertion (74-113 aa). Particularly, 1Slx-129 had an extra cysteine residue at the position 109 due to a TAT → TGT dot mutation, which may improve the formation of superior gluten macropolymer. Our results suggest that these unusual HMW-GS could be served as potential superior gene resources for improving wheat gluten quality. Phylogenetic analysis revealed that HMW-GS genes from Glu-1Sx genomes had close evolutionary relationships with those of Glu-1Dx genome while sequences from Ae. speltoides aligned with those of B genome.