Variants of the 350-family sequences were isolated from Dasypyrum villosum genomic DNA. Although the consensus sequence shared 86% homology to p380 and 84% homology to pHvNAU62 of D. villosum, a 63 bp long region including variable microsatellite repeat (TG)(7-9) was found to be absent in pHvNAU62 or much different from p380. This sequence also shared 67% homology to the 350-family of rye. The representative clone pDvTU383 was localized on the D. villosum chromosomes by using the sequential C-banding and fluorescence in situ hybridization (FISH). Combining with the probes of pTa71 and pTa794 containing 18S-5.8S-28S rDNA (NOR) and 5S rDNA multigene families, respectively, the pDvTU383 probe allowed identification of all seven pairs of D. villosum chromosomes. The pair of the 1V chromosomes fluoresced strong in situ hybridization signals of 18S-5.8S-28S rRNA genes in the NORs of 1VS, and the pair of the 5V chromosomes had weak signals of 5S rRNA genes at the end of 5VS. The pair of the 4V chromosomes showed sub-telomeric signals of pDvTU383 probe on the both arms, however, the signal of the short arm is weaker than that of the long arm. The pair of the 7V chromosomes showed no signals of pDvTU383 on the both sub-telomeric regions. The pair of the 6V chromosomes showed a characteristic interstitial signal of pDvTU383 on the short arm. The pairs of the 2V and 3V chromosomes were distinguished by the difference of interstitial signals of pDvTU383 on their long arms. Furthermore, hybridization signals of pDvTU383 were also visualized on centromeres of all chromosomes. Based on the above D. villosum chromosomal patterns made by FISH, one pair of the 4V chromosomes originated from D. villosum were found in a trigeneric hybrid line involving wheat, Dasypyrum and Thinopyrum. The sequence pDvTU383 provides an effective probe for further analysis of the D. villosum genome.