Abstract: Homeodomain-leucine zipper proteins are unique to plants and regulate many aspects of plant development as well as plant tolerance to abiotic stress. However, the function of the most family members is largely unknown. Molecular studies of tall fescue lag behind other model species even though this plant is widely used as a cool-season turf grass. A local nucleotide database file was created in Bioedit software based on published and our own unpublished tall fescue transcriptome data. Using 14 HD-Zip I protein sequences of rice as the query, 13 counterparts in tall fescue were obtained by the Tblastn search program. Based on these obtained sequences, a full length open reading frame of the 13 FaHD-Zip I was cloned by nest PCR method. Phylogenetic and MEME motif analyses showed that all 13 FaHD-Zip I were closely related to the corresponding orthologs in rice. In addition, the number and arrangement mode of conserved motifs was nearly the same in the two species. qRT-PCR was adopted to analyze the expression pattern of 11 FaHD-Zip I in crowns under polyethylene 6000 (PEG6000) treatment. The relative expression level of FaHD-Zip I genes (except FaHOX4 and FaHOX6) were up- or down-regulated under short-term PEG6000 treatment, with FaHOX22 the most significantly up-regulated. All gene expression was induced in crowns of PEG6000-treated plants compared with that in control plants at 7 or 14 d. Relative expression level of FaHOX22 was up-regulated by 76.3 fold. This study provides a fast and efficient method for molecular cloning of certain transcription factor family members in species that lack genomic sequences. Furthermore, it promotes the future investigation of the molecular function of key factors that may serve a useful role in molecular breeding.