Two DNA markers, i.e., a fragment mixture (Thg-m) and a Thinopyrum intermedium-enriched moderate-repetitive sequence (Thg36) were obtained by microdissection of a pair of added chromosomes in a wheat-Th. intermedium disomic addition line (TAI-27), followed by direct cloning of the amplification product. The amplification product was produced by two sequential PCR amplifications: first by degenerate oligonucletide-primed PCR (DOP-PCR) on the dissected chromosomes as a template; and second by sequence-specific PCR using primers designed according to highly conserved domains between two plant disease-resistance genes. Genomic Southern blot hybridization on relevant plant materials, including partial amphiploid, addition line, putative translocation line as well as parental lines, showed that the two DNA markers are useful in detecting Th. intermedium chromatin introgressed into the wheat genome. Allocation of two low-copy sequences cloned from the same source to individual wheat chromosomes confirmed the origin of the microdissected target chromosome.