Metabolic changes occur in mammary gland cells of dairy cows with negative energy balance may alter milk fatty acid profile. The aim of current study is to assess the concentrations of fatty acids in milk of dairy cows exploring gas chromatography- mass spectrophotometry (GC-MS) analysis. Twenty milk samples had collected from ten dairy cows, two samples from each cow 3w prepartum and 3w postpartum. All samples were lyophilized and then analyzed using GC-MS to extinguish their fatty acid metabolites. The results of this study revealed that palmitic acid (C16:0) is the prominent fatty acid in cow milk with a higher percentage of fatty acids in all groups of the study followed by oleic acid (C18:1 cis-9), stearic acid (C18:0), and myristic acid (C14:0), respectively. Except in normal-status cow milk prepartum which have no differentiated GC-MS stearic acid. In addition, palmitic acid decreased in NEB compared to normal-status cow milk both pre- and postpartum. Oleic acid and stearic acid decreased in NEB compared to normal-status group postpartum only. While myristic acid increased both pre- and postpartum in NEB group compared to normal-status cows. However, all these differences in fatty acid concentrations did not reach significant levels (p>0.05). We can conclude that NEB-associated metabolic disturbances in lipolysis and lipogenesis lead to alterations in cow milk fatty acid profile and the long-chain fatty acids are the more affected ones, both pre- and postpartum.