Hv-S/TPK gene, a resistance related gene to powdery mildew, was cloned by using genechip, and its expression was upregulated after the inoculation of Blumeria graminis to Haynaldia villosa. Using the specific primers of Hv-S/TPK to screen a genomic TAC (Transformation-competent artificial chromosome) library of translocation line 6VS/6AL, a positive TAC was screened. A 5-kb fragment containing Hv-S/TPK was subcloned and identified. This 5160-bp fragment (GenBank Accession No. EU153366) was determined by specific primer walking. The analysis of Hv-S/TPK genomic sequence showed three introns and four extrons between start code and stop code. In the promoter region of Hv-S/TPK, there were W-box and OCS-like elements which were the elements related to disease resistance. In this study, the positive TAC clone was used to as probe in situ hybridized to mitotic metaphase chromosomes of translocation line. The result of fluorescence in situ hybridization (FISH) indicated that the TAC clone containing Hv-S/TPK was from Haynaldia villosa chromosome.