Bovine milk fat globules naturally vary from less than 0.2 µm to 15 µm in diameter. Milk has at least two distinct distributions of fat globules. While the majority (~90%) of globules in milk are of the smaller distribution (average diameter of 0.4 µm), virtually all the fat is carried in the larger globules (average diameter 3.5 µm). This distribution suggests some compositional and/or functional significance might exist between the two populations of fat globules, which may be related to origin of these globules in the lactating cell. Milk fat globules have a unique structure, composed of a core droplet of non polar lipids (triacylglycerol) surrounded by a lipid bilayer membrane known as milk fat globule membrane (MFGM). Other than MFGM, there is another source of membrane that has been identified in skim milk. It has been hypothesized that this skim milk membrane (SMM) is derived from MFGM, but little data are available to support this idea, and the membrane may also have alternate origins. In this study, different aggregates of lipids (small and large fat globules, SMM, skim milk) from milk were isolated and characterized for their lipid contents. Isolation of small and large fat globules fractions was verified by laser diffraction particle size analysis. The lipids were extracted from isolated different lipid aggregates and individual classes were separated using thin layer chromatography. Lipids were transesterified to fatty acid methyl esters and analyzed by gas chromatography-mass spectrometry. The results indicate that there are some compositional differences between native milk fat globule membranes of different sizes. For example, the total phospholipid fraction of small fat globules (SFG) contained significantly more unsaturated C18:1n9 and C18:2n6 than large fat globules (LFG). Conversely, sphingomyelin composition of SFG contained less C18:1n9 and C18:2n6cc, but more long chain fatty acids C22:0, C23:0, and C24:0. Phosphatidylethanolamine composition of SMM contained more C17:1 than SFG and LFG. The composition of C18:1n9 in triacylglycerol increased with fat globule size. Clear differences were also found in lipid profile of SMM and small and large fat globules from milk. Composition differences between SMM and native milk fat globules of different sizes suggest that origin of this membrane material in skim milk might have some different source than that of MFGM.