Abstract HSP70/90 play important role in testis develop and spermatozoa regulation, but the contact of HSP70/90 with infertility in cattle is unclear. Here, we focus on male cattle-yak and yak, which to investigate the expression and localization of HSP70/90 in different tissues, and explore the influence of HSP70/90 to infertility. In our study, a total of 54 cattle (24 cattle-yak and 30 yak) were examined. The HSP90 mRNA of cattle-yak was cloned first and found amino acid variation in HSP90, which led to difference at protein spatial structure compare with yak. To investigate whether the expression of HSP70/90 mRNA and protein are different in cattle-yak and yak, we used real-time quantitative PCR (qRT-PCR) and Western blot (WB) to examine them. We found that the expression level of HSP70/90 mRNA and protein are disparity in different tissues and testis development stages, and obviously high expression was observed in testicle during juvenile and adult, Moreover, it‘s interestingly in which the HSP70 expression is significant high in yak whereas HSP90 in cattle-yak (P<0.01). On this bases, we detect the location of HSP70/90 in testis by immunohistochemical (IHC) and immunofluorescence (IF), the results demonstrate that HSP70/90 were located in the epithelial cells, spermatogenic cells and mesenchymal cells. In summary, our study proved the expression of HSP70/90 are different in tissues, and the expression of HSP90 is obviously high in testis of cattle-yak, which propose that the infertility of cattle-yak may cause from up-regulating of HSP90.