Heat shock protein 27 (Hsp27)/protein 53 (P53) plays an important role in testis development and spermatozoa regulation, but the relationship between Hsp27/P53 and infertility in cattle is unclear. Here, we focus on male cattle‐yak and yak to investigate the expression and localization of Hsp27/P53 in testis tissues and to explore the influence of Hsp27/P53 on infertility. In our study, a total of 54 cattle (24 cattle‐yak and 30 yak) were examined. The Hsp27 and P53 messenger RNA (mRNA) of cattle‐yak were cloned, and amino acid variations in Hsp27 and P53 were found; the variations led to differences in the protein spatial structure compared with yak. We used real‐time quantitative polymerase chain reaction and western blot to investigate whether the expression of Hsp27/P53 mRNA and protein was different in cattle‐yak and yak. We found that the expression levels of Hsp27/P53 mRNA and protein were different in the testis developmental stages and the highest expression was observed in testicles during adulthood. Moreover, the Hsp27 expression was significantly higher in yak, whereas P53 expression was higher in cattle‐yak ( p < 0.01). On this basis, we detected the location of Hsp27/P53 in the testis by immunohistochemistry and immunofluorescence. The results demonstrated that Hsp27 was located in spermatogenic cells at different developmental stages and mesenchymal cells of the yak testicles. However, P53 was located in the primary spermatocyte and interstitial cells of the cattle‐yak testicles. In summary, our study proved that the expression of Hsp27/P53 differed across the testis developmental stages and the expression of P53 was higher in the testis of cattle‐yak, which suggested that the infertility of cattle‐yak may be caused by the upregulation of P53.