Milk has been an issue of public health concern due to unhealthy fats. This study investigated changes in milk fatty acids (FA) in relation to blood metabolites with advancing age and lactation stage in dairy buffaloes. A total of 12 dairy buffaloes were studied for six weeks in Peshawar. The serum glucose concentration averaged 59.02 mg/dl and triglyceride were 54.63 mg/dl. Glucose and triglyceride concentration was a bit high in older animals. Saturated FAs (SFAs) were averaged 70.41 g/100g. C14:1, C16:1 and C18:3 were significantly (P<0.05) higher in younger buffaloes while C18:1cis, medium chain FA and long chain FA were higher in older ones (P <0.05). Desaturase activity was significantly (P <0.05) higher in younger animals. Even though the SFA and UFA were not significantly affected by age but the data trend showed a little higher SFA in older animals compared to younger ones. The ratio of ?9 desaturase activity was also non-significantly affected by lactation stage. It was concluded that blood glucose and triglyceride were lower, milk FA quality was better and desaturase was higher in younger animals at early lactation.