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Among the established breeds of buffalo in Pakistan, the Nili-Ravi breed is dominant in the Punjab Province. The purpose of the study is to determine the chemical composition and fatty acid profile of buffalo milk. Milk is a complete food and contains all the nutritional components in balanced form. It helps to meet important nutritional needs of the human body. The buffaloes were divided into 3 groups on the basis of lactation period i.e. early, middle and late lactation. A total of 90 milk samples were obtained for analysis. Buffalo milk samples were analyzed for proximate analysis, parameters like fat, crude protein, lactose, ash, solids not fat (SNF), total solids (TS), pH and acidity were determined. Fatty acid profile of buffalo milk was determined by the method, Association of Office of Agricultural Chemists (AOAC, 2000). Milk fat content (%) was found to be significantly (p<0.05) lower in the early (6.52) and mid lactation (6.61) stages than the late lactation stage (6.85). The content of TS (%) was found to be significantly (p<0.05) higher in the late lactation (14.62 and 15.45) stage than the early (12.94 and 13.65)) and mid lactation (13.76 and 14.14) stages. In contrast, the content of total protein (4.31% to 4.78%), Solid not fat (8.40% to 8.70%), lactose (4.36% to 4.74%) and ash (0.18% to 0.19%) did not vary significantly among the different lactation stages. The values of Butyric acid, Caproic acid, Myristic acid, Stearic acid and Oleic acid showed significant increase towards end of lactation. These values of fatty acids also revealed that there is significant difference among the animals and lactation stages of Nili Ravi buffalo milk. Results showed that Capric acid, Lauric acid, Palmitic acid, Linoleic acid and Linolenic acid decreased in early to middle and increased towards end of lactation. [ABSTRACT FROM AUTHOR]