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Thinopyrum intermedium is an important species with potential utilization value in breeding of wheat. In this study, the non-coding intergenic region of trnH-psbA was investigated to assess the genetic diversity and infer the maternal origin within T. intermedium accessions. Eleven haplotypes were distinguished among the thirty-five accessions of T. intermedium. They showed a relatively low nucleotide diversity (π) of 0.00473 ± 0.00037 and a moderately high haplotype diversity (Hd) of 0.733 ± 0.061. In the phylogenetic analysis, all accessions of T. intermedium were positioned into two clades, which corresponded to the different diploid donors. These results suggested that there were two phylogenetically divergent maternal donors in T. intermedium.