Heat shock factor binding protein 1(HSBP1) is a nuclear-localized, novel, conserved, low molecular weight (< 100 residues) transcriptional factor, which may repress the activity of the heat shock factor 1 (HSF1) by binding HSF1 active trimerization domain. HSBP1 gene have been cloned in human and mouse, but not reported in rat. In this paper, a pair of consensus degenerate primers were designed based on N-terminal and C-terminal conservative amino acid sequence. Using RT-PCR method, hsbp1 gene fragment was amplified and cloned from total RNA extracted from rat C6 glioma cells. Then the EST was probed to isolate the rat full-length hsbp1 cDNA by in situ hybridization from a rat C6 glioma cells cDNA library. The full-length hsbp1 was deposited in GenBank (accession No. AY522937). It was blasted in RGD (rat genome database) and was localized in 19q12 and composed of four extrons and three introns. The distance between the first extron and the fourth extron was 5829bp. Then its Uinigene was searched, results showed HSBP1 existed widely in all kinds of organs and tissues, the data suggested that it may play a important roles in physiological activity. In addition, the sequence similarity and phylogenetic relationship were compared with DNAman tool. The result showed the relationship is consistent between the similarity of amino acid sequence and phylogenetic evolution from morphological of those species which were nearly in evolution.