Yak milk, known as the “liquid gold”, is a nutritious food with extensive consumption. Compared with cow milk, yak milk contains higher levels of nutrients such as dry matter, milk fat, and milk protein, which demonstrates great potential for exploitation and utilization. Protein kinase cGMP-dependent 1 (PRKG1) is an important functional molecule in the cGMP signaling pathway, and its significant influence on milk fatty acids has been discovered. The aim of this study is to explore the correlation between single nucleotide polymorphisms (SNPs) in the PRKG1 gene and the quality traits of Gannan yak milk in order to identify candidate molecular markers for Gannan yak breeding. In this study, genotyping was performed on 172 healthy, 4–5-year-old lactating Gannan yaks with similar body types, naturally grazed, and two to three parity. Three SNPs (g.404195C>T, g.404213C>T, and g.760138T>C) were detected in the PRKG1 gene of Gannan yaks, which were uniformly distributed in the yak population. Linkage disequilibrium analysis was conducted, revealing complete linkage disequilibrium between g.404195C>T and g.404213C>T. After conducting a correlation analysis between SNPs in the PRKG1 gene and milk quality in Gannan yaks, we found that PRKG1 SNPs significantly increased the content of casein, protein, and SNFs in yak milk. Among them, the TT homozygous genotype at the PRKG1 g.404195C>T loci exhibited higher casein and protein contents compared to the CC and CT genotypes (p < 0.05). The SNP g.760138T>C locus was associated with casein, protein, SNFs, and TS traits (p < 0.05). The CC genotype had higher casein and protein contents than the TT and TA genotypes (p < 0.05). However, there were no significant differences in milk fat, lactose, and acidity among the three genotypes (p > 0.05). In summary, PRKG1 gene polymorphism can serve as a candidate molecular marker for improving milk quality in Gannan yaks.