Cattle-yak, the hybrid offspring of yak (Bos grunniens) and cattle (Bos taurus), serves as a unique model to dissect the molecular mechanisms underlying reproductive isolation. While female cattle-yaks are fertile, the males are completely sterile due to spermatogenic arrest at the meiosis stage and massive germ cell apoptosis. Interestingly, meiotic defects are partially rescued in the testes of backcrossed offspring. The genetic basis of meiotic defects in male cattle-yak remains unclear. Structure-specific endonuclease subunit (SLX4) participates in meiotic double-strand break (DSB) formation in mice, and its deletion results in defects in spermatogenesis. In the present study, we examined the expression patterns of SLX4 in the testes of yak, cattle-yak, and backcrossed offspring to investigate its potential roles in hybrid sterility. The results showed that the relative abundances of SLX4 mRNA and protein were significantly reduced in the testis of cattle-yak. The results of immunohistochemistry revealed that SLX4 was predominately expressed in spermatogonia and spermatocytes. Chromosome spreading experiments showed that SLX4 was significantly decreased in the pachytene spermatocytes of cattle-yak compared with yak and backcrossed offspring. These findings suggest that SLX4 expression was dysregulated in the testis of cattle-yak, potentially resulting in the failure of crossover formation and collapses of meiosis in hybrid males.