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This study aimed to investigate the spatially and temporally expressed patterns and biological characteristics of <i>TSSK1B</i> in male yaks and explore the potential correlation between <i>TSSK1B</i> and male sterility of the yak hybrid offspring (termed cattle–yak). First, the coding sequence (CDS) of <i>TSSK1B</i> was cloned by RT-PCR, and bioinformatics analysis was conducted with relevant software. Quantitative real-time PCR (RT-qPCR) was employed to detect the expression profile of <i>TSSK1B</i> in various tissues of male adult yaks, the spatiotemporal expression of <i>TSSK1B</i> in different stages of yak testes, and the differential expression of <i>TSSK1B</i> between yak and cattle–yak testes. The cellular localization of <i>TSSK1B</i> was determined by immunohistochemistry (IHC). Furthermore, the methylation status of the <i>TSSK1B</i> promoter region was analyzed by bisulfite-sequencing PCR (BSP). The results showed that <i>TSSK1B</i> was 1235 bp long, including 1104 bp of the CDS region, which encoded 367 amino acids. It was a conserved gene sharing the highest homology with <i>Bos mutus</i> (99.67%). In addition, the bioinformatics analysis revealed that TSSK1B was an unstable hydrophilic protein mainly containing the alpha helix of 34.06% and a random coil of 44.41%, with a transmembrane structure of 29 amino acids long. The RT-qPCR results demonstrated that <i>TSSK1B</i> was specifically expressed in yak testes compared with that in other tissues and especially highly expressed in adult yak testes. On the contrary, <i>TSSK1B</i> was hardly expressed in the testis of adult cattle–yak. IHC confirmed that <i>TSSK1B</i> protein was more strongly expressed in the testes of adult yaks than in their fetal and juvenile counterparts. Interestingly, nearly no expression was observed in the testes of cattle–yak compared with the corresponding testes of yak. Bisulfite-sequencing PCR (BSP) revealed that the methylated CpG sites in the <i>TSSK1B</i> promoter region of cattle–yak was significantly higher than that in the yak. Taken together, this study revealed that <i>TSSK1B</i> was specifically expressed in yak testes and highly expressed upon sexual maturity. Moreover, the rare expression in cattle–yak may be related to the hypermethylation of the promoter region, thereby providing a basis for further studies on the regulatory mechanism of <i>TSSK1B</i> in male cattle–yak sterility.