Milk fat is a complex natural fat and contains around 400 fatty acids. The objectives of this study were to extract fat from bovine milk using two different methods, including Bligh and Dyer and Mojonnier, and to determine the fatty acid content in the extracted fats using gas chromatography (GC). No differences (p > .05) were detected in the fat content and fatty acids content as a percentage of total fat (FA%TF) extracted using both methods. No differences (p > .05) were detected in some saturated fatty acids (SFAs) and unsaturated fatty acids (USFAs) extracted from both methods, such as C11:0 (undecylic acid), C16:0 (palmitic acid), C18:0 (stearic acid), C14:1 (myristoleic acid), and C16:1 (palmitoleic acid). However, the majority of SFAs were different (p < .05) in Mojonnier method as compared to Bligh and Dyer method and vice versa for USFAs. The short (6.54% vs. 5.95%) and medium (21.86% vs. 20.73%) chains FAs determined by GC were high in Mojonnier fat as compared to Bligh and Dyer fat, while the long-chain FAs were higher in the last (66.61%) relative to Mojonnier fat (65.51%). This study found that Mojonneir method has resulted in fewer errors. In contrast, the Bligh and Dyer extraction method has more experimental error, which led to decreasing the total fat, as well as was not able to detect C9:0.