Milk quality has been an issue of public health concern and changes occur in milk composition with the changing physiological and management states. An experiment was conducted to investigate changes in milk yield and fatty acids profile with advancing age and lactation stage in dairy buffaloes. A total of 12 multiparous lactating dairy buffaloes were divided into two groups younger (1-4 lactation number) and mature (8-10 lactation number) having six animals in each one, which were further subdivided into early (1-3 months) and late (7-10 months) lactation stage. All the buffaloes were stall fed, green fodders were provided ad libitum and concentrate mixture at the scale of 1 kg/2 lit of milk production. The experiment continued for six weeks starting from November upto mid of December 2009. Milk samples were weekly collected for analysis of fatty acids profile. Saturated FAs (SFAs) showed the highest concentration out of the total milk fatty acids and averaged 70.41 g/100 g ranging from 64.96 to 78.83 g/100 g. The concentration of C14:1, C16:1 and C18:3 was significantly (P<0.05) higher in younger buffaloes while C18:1cis, medium chain fatty acids (MCFA) and long chain fatty acids (LCFA) were higher in older ones (P <0.05). The ratio of Δ9 desaturase activity was significantly (P <0.05) higher in younger animals (0.11) compared to older (0.07) while it was not affected by lactation stage. It was concluded that the milk fatty acids quality was better in younger animals at early lactation.