Yak milk is superior to common cow milk in nutrients including protein, fat and calories. However, the milk yield of the yak is very much lower compared with other dairy bovines. To understand the molecular mechanisms of lactogenesis, lactation and mammary gland development, mammary tissue samples were taken from five yaks during a dry period (DP, n = 3) and lactation period (LP, n = 2). Two types of cDNA sequence libraries that reflected the different physiological states of the mammary gland were constructed using RNA sequencing technology. After removing reads containing adapters, reads containing poly-N and low-quality reads from the raw data, 45,423,478 to 53,274,976 clean reads were obtained from these libraries. A total of 74.72% to 80.65% of the high-quality sequence reads were uniquely aligned to the BosGru v2.0 yak reference genome. Using the DESeq R package, 360 differentially expressed genes were detected between the two groups when the adjusted P value (padj < 0.05) was used as the cutoff value; this included 192 upregulated and 168 downregulated genes in the yak mammary gland tissue of the DP compared to the LP. A gene ontology analysis revealed that the most enriched GO terms were protein binding, multi-organism process, immune system and others. KEGG pathway analysis indicated that the differentially expressed genes were mostly enriched in Hippo signaling, insulin signaling, steroid biosynthesis and others. The analysis of the up- and downregulated genes provides important insights into the molecular events involved in lactogenesis, lactation and mammary gland development and will guide further research to enhance milk yield and optimize the constituents of yak milk.