Yak (Bos grunniens) is a unique bovine species and considered as lifeline of highlanders. The male subfertility in yak is a matter of concern that causes huge economic loses. The spermatogenesis and male reproduction machinery are critically governed by Y-linked genes which tend to acquire necessary information in the course of evolution. The Y-linked fertility genes are present in multiple copies with testis-limited expression. To understand this novel complexity, 12 male-specific region of Y chromosome (MSY) genes have been studied in the yak. Targeted genes are amplified in male and female genomic DNA and confirmed the male derived specificity. Moreover, testis and sperm-specific expressions of MSY genes are distinct among different tissues. The quantitative polymerase chain reaction results validate the expression pattern of these genes in various tissues with predominant expression intestis and sperm. The sequencing of resultant yak MSY genes gives significant result and shows similarity with cattle (Bos indicus), but few nucleotide mismatches define the proposition of infertile male in the F₁ hybrid of cattle and yak. The identified MSY genes can be used to establish male-specific characteristics and to differentiate male and female yak genotypically. Further, these genes may act as valuable resources to understand the capacity of spermatogenesis, embryogenesis, cellular growth, azoospermia and malesubfertility in the yak.