In recent years, milk fat has garnered significant attention. Over 98% of milk fat is composed of triglycerides, with fatty acids being one of the main components that play important physiological roles. The distribution of fatty acids on the glycerol backbone of triglycerides is closely related to the digestion and absorption of milk fat. However, there is a lack of research comparing the stereospecific distribution of fatty acids in human milk and different ruminant milks. This review summarizes the extraction techniques and analytical methods of fatty acids from milk fat, analyzes and compares the composition and content of total fatty acids in human, yak, camel, cow, buffalo, and goat milks, overviews the stereospecific location of fatty acids on triglycerides and digestion and absorption of fatty acids in different milk sources, and evaluates the nutritional value of different milk sources using seven fatty acid nutritional evaluation indices to evaluate the nutritional value of different milk sources, providing guidance for promoting human health, and exploring the potential importance of the fatty acid composition of ruminant milk for infant or special dairy development, providing a basis for further development and utilization of dairy products.