Dasypyrum villosum is a valuable genetic resource for wheat improvement. With the aim to efficiently monitor the D. villosum chromatin introduced into common wheat, two novel retrotransposon sequences were isolated by RAPD, and were successfully converted to D. villosum-specific SCAR markers. In addition, we constructed a chromosomal karyotype of D. villosum. Our results revealed that different accessions of D. villosum showed slightly different signal patterns, indicating that distribution of repeats did not diverge significantly among D. villosum accessions. The two SCAR markers and FISH karyotype of D. villosum could be used for efficient and precise identification of D. villosum chromatin in wheat breeding.