Cow milk is rich in proteins, fats, carbohydrates, and minerals; however, its precise nutrient content varies based on various factors. In the current study, we evaluated the differences in the fatty acid and protein contents of milk and the factors associated with these differences. To achieve this, samples were collected from seven types of cows in different regions. These included samples from three dairy breeds: Chinese Holstein milk from Beijing, China (BH), Chinese Holstein milk (HH) and Jersey milk (JS) from Hebei province, China; and four dairy/meat breeds: Sanhe milk (SH) from Inner Mongolia Autonomous Region, China, Xinjiang brown milk (XH) and Simmental milk (SI) from Xinjiang Uygur Autonomous Region, China, and Shu Xuanhua milk (SX) from Sichuan province, China. Breed significantly affects total fat, fatty acid, and protein contents. Additionally, geographic region significantly affects the contents of different fatty acids, α-lactalbumin, and lactoferrin. JS has the highest total fat and casein contents. XH samples contain significantly higher unsaturated fatty acid content than BH samples and do not differ significantly from JS. Additionally, the low β-lactoglobulin and high lactoferrin contents in XH samples may be favorable for the growth and development of infants. Our results may inform the development of dairy products from different cow breeds and advance the process of accurate breed identification.